Methods, Cells &amp; Organisms

ABSTRACT

The invention relates to an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (e.g., in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location. The technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (e.g., produced by NHEJ) in the present invention and thus is more efficient than prior art techniques. The invention also provides sequential insertion and/or deletions using single- or double-stranded DNA cutting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 ofco-pending International Application No. PCT/GB2014/052837 filed Sep.18, 2014, which designated the U.S., and which claims benefit of GBApplication No. 1316560.0 filed Sep. 18, 2013, and claims benefit of GBApplication No. 1321210.5 filed Dec. 2, 2013 the contents of each ofwhich are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 11, 2016, isnamed K00016-1-Sequence-Listing-069496-086061.txt and is 1,444,445 bytesin size.

The inventors have devised an approach for introducing one or moredesired insertions and/or deletions of known sizes into one or morepredefined locations in a nucleic acid (e.g., in a cell or organismgenome). They developed techniques to do this either in a sequentialfashion or by inserting a discrete DNA fragment of defined size into thegenome precisely in a predefined location or carrying out a discretedeletion of a defined size at a precise location. The technique is basedon the observation that DNA single-stranded breaks are preferentiallyrepaired through the HDR pathway, and this reduces the chances of indels(e.g., produced by NHEJ) in the present invention and thus is moreefficient than prior art techniques.

The inventors have also devised new techniques termed sequentialendonuclease-mediated homology directed recombination (sEHDR) andsequential Cas-mediated homology directed recombination (sCHDR).

BACKGROUND

Certain bacterial and archaea strains have been shown to contain highlyevolved adaptive immune defence systems, CRISPR/Cas systems, whichcontinually undergo reprogramming to direct degradation of complementarysequences present within invading viral or plasmid DNA. Short segmentsof foreign DNA, called spacers, are incorporated into the genome betweenCRISPR repeats, and serve as a ‘memory’ of past exposures. CRISPRspacers are then used to recognize and silence exogenous geneticelements in a manner analogous to RNAi in eukaryotic organisms.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)system including the CRISPR associated (Cas) protein has beenreconstituted in vitro by a number of research groups allowing for theDNA cleavage of almost any DNA template without the caveat of searchingfor the right restriction enzyme cutter. The CRISPR/Cas system alsooffers a blunt end cleavage creating a dsDNA or, using mutated Casversions, a selective single strand-specific cleavage (see Cong et al.,Wang et al. & Mali et al. cited below).

Through in vitro studies using Streptococcus pyogenes type II CRISPR/Cassystem it has been shown that the only components required for efficientCRISPR/Cas-mediated target DNA or genome modification are a Cas nuclease(e.g., a Cas9 nuclease), CRISPR RNA (crRNA) and trans-activating crRNA(tracrRNA). The wild-type mechanism of CRISPR/Cas-mediated DNA cleavageoccurs via several steps. Transcription of the CRISPR array, containingsmall fragments (20-30 base-pairs) of the encountered (or target) DNA,into pre-crRNA, which undergoes maturation through the hybridisationwith tracrRNA via direct repeats of pre-crRNA. The hybridisation of thepre-crRNA and tracrRNA, known as guide RNA (gRNA or sgRNA), associateswith the Cas nuclease forming a ribonucleoprotein complex, whichmediates conversion of pre-crRNA into mature crRNA. MaturecrRNA:tracrRNA duplex directs Cas9 to the DNA target consisting of theprotospacer and the requisite protospacer adjacent motif (CRISPR/casprotospacer-adjacent motif; PAM) via heteroduplex formation between thespacer region of the crRNA and the protospacer DNA on the host genome.The Cas9 nuclease mediates cleavage of the target DNA upstream of PAM tocreate a double-stranded break within the protospacer or astrand-specific nick using mutated Cas9 nuclease whereby one DNAstrand-specific cleavage motif is mutated (for example, Cas9 nickasecontains a D10A substitution) (Cong et al.).

It is worth noting that different strains of Streptococcus have beenisolated which use PAM sequences that are different from that used byStreptococcus pyogenes Cas9. The latter requires a NGG PAM sequence.CRISPR/Cas systems (for example, the Csy4 endoribonulcease inPseudomonas aeroginosa (see Shah et al.) have been described in otherprokaryotic species, which recognise a different PAM sequence (e.g.,CCN, TCN, TTC, AWG, CC, NNAGNN, NGG, NGGNG). It is noteworthy that theCsy4 (also known as Cas6f) has no sequence homology to Cas9 but the DNAcleavage occurs through a similar mechanism involving the assembly of aCas-protein-crRNA complex that facilitates target DNA recognitionleading to specific DNA cleavage (Haurwitz et al.).

In vitro-reconstituted type II CRISPR/Cas system has been adapted andapplied in a number of different settings. These include creatingselective gene disruption in single or multiple genes in ES cells andalso single or multiple gene disruption using a one-step approach usingzygotes to generate biallelic mutations in mice. The speed, accuracy andthe efficiency at which this system could be applied to genome editingin addition to its multiplexing capability makes this system vastlysuperior to its predecessor genome editing technologies namely zincfinger nucleases (ZFNs), transcription activator-like effector nucleases(TALENs) and engineered homing meganucleases (Gaj et al. & Perez-Pineraet al.). These have been successfully used in various eukaryotic hostsbut they all suffer from important limitations; notably off-targetmutagenesis leading to nuclease-related toxicity, and also the time andcost of developing such engineered proteins. The CRISPR/Cas system onthe other hand is a superior genome editing system by which mutationscan be introduced with relative ease, simply by designing a singleguided RNA complementary to the protospacer sequence on the target DNA.

The dsDNA break induced by an endonuclease, such as Cas9, issubsequently repaired through non-homologous end joining mechanism(NHEJ), whereby the subsequent DNA repair at the breakpoint junction isstitched together with different and unpredictable inserted or deletions(indels) of varying size. This is highly undesirable when precisenucleic acid or genome editing is required. However a predefined precisemutation can be generated using homology directed repair (HDR), e.g.,with the inclusion of a donor oligo or donor DNA fragment. This approachwith Cas9 nuclease has been shown to generate precise predefinedmutations but the efficiency at which this occurs in both alleles is lowand mutation is seen in one of the strands of the dsDNA target (Wang etal.).

The CRISPR/Cas system does therefore have some limitations in itscurrent form. While it may be possible to modify a desired sequence inone strand of dsDNA, the sequence in the other strand is often mutatedthrough undesirable NHEJ.

SUMMARY OF THE INVENTION

A first configuration of the present invention provides:—

A method of nucleic acid recombination, the method comprising providingdsDNA comprising first and second strands and

-   -   (a) using nucleic acid cleavage to create 5′ and 3′ cut ends in        the first strand;    -   (b) using homologous recombination to insert a nucleotide        sequence between the ends, thereby producing a modified first        strand; thereby producing DNA wherein the first strand has been        modified by said recombination but the second strand has not        been modified; and    -   (c) optionally replicating the modified first strand to produce        a progeny dsDNA wherein each strand thereof comprises a copy of        the inserted nucleotide sequence; and isolating the progeny        dsDNA.        A second configuration of the present invention provides:—

A method of nucleic acid recombination, the method comprising

-   -   (a) using nucleic acid cleavage to create 5′ and 3′ cut ends in        a single nucleic acid strand;    -   (b) using homologous recombination to insert a nucleotide        sequence between the ends, wherein the insert sequence comprises        a regulatory element or encodes all or part of a protein; and    -   (c) optionally obtaining the nucleic acid strand modified in        step (b) or a progeny nucleic strand comprising the inserted        nucleotide sequence.        A third configuration of the present invention provides:—

A method of nucleic acid recombination, the method comprising

-   -   (a) using nucleic acid cleavage to create first and second        breaks in a nucleic acid strand, thereby creating 5′ and 3′ cut        ends and a nucleotide sequence between the ends;    -   (b) using homologous recombination to delete the nucleotide        sequence; and    -   (c) optionally obtaining the nucleic acid strand modified in        step (b) or a progeny nucleic strand comprising the deletion.        A fourth configuration of the present invention provides:—

A method of nucleic acid recombination, the method comprising providingdsDNA comprising first and second strands and

-   -   (a) using Cas endonuclease-mediated nucleic acid cleavage to        create a cut end in the first strand 3′ of a PAM motif;    -   (b) using Cas endonuclease-mediated nucleic acid cleavage to        create a cut in the second strand at a position which        corresponds to a position 3′ of the cut end of the strand of        part (a), which cut is 3′ of the PAM motif;    -   (c) providing a first gRNA which hybridises with a sequence 5′        to the PAM motif in the strand of part (a)    -   (d) providing a second gRNA which hybridises with a sequence 5′        to the PAM motif in the strand of part (b)        -   wherein the nucleic acid strands of part (a) and part (b)            are repaired to produce a deletion of nucleic acid between            the cuts.

In aspects of the configurations of the invention there is provided amethod of sequential endonuclease-mediated homology directedrecombination (sEHDR) comprising carrying out the method of anypreceding configuration a first time and carrying out the method of anypreceding configuration a second time. In this way, the inventionenables serial nucleic acid modifications, e.g., genome modifications,to be carried out, which may comprise precise sequence deletions,insertions or combinations of these two or more times. For example, itis possible to use this aspect of the invention to “walk along” nucleicacids (e.g., chromosomes in cells) to make relatively large and precisenucleotide sequence deletions or insertions. In an embodiment, one ormore Cas endonucleases (e.g., a Cas9 and/or Cys4) is used in a method ofsequential Cas-mediated homology directed recombination (sCHDR).

In another aspect, the invention can be described according to thenumbered sentences below:

-   -   1. A method of nucleic acid recombination, the method comprising        providing dsDNA comprising first and second strands and    -   (a) using nucleic acid cleavage to create 5′ and 3′ cut ends in        the first strand;    -   (b) using homologous recombination to insert a nucleotide        sequence between the ends, thereby producing a modified first        strand; thereby producing DNA wherein the first strand has been        modified by said recombination but the second strand has not        been modified; and    -   (c) optionally replicating the modified first strand to produce        a progeny dsDNA wherein each strand thereof comprises a copy of        the inserted nucleotide sequence; and isolating the progeny        dsDNA.

2. A method of nucleic acid recombination, the method comprising

-   -   (a) using nucleic acid cleavage to create 5′ and 3′ cut ends in        a single nucleic acid strand;    -   (b) using homologous recombination to insert a nucleotide        sequence between the ends, wherein the insert sequence comprises        a regulatory element or encodes all or part of a protein; and    -   (c) optionally obtaining the nucleic acid strand modified in        step (b) or a progeny nucleic strand comprising the inserted        nucleotide sequence.

3. The method of any preceding sentence, wherein the insert sequencereplaces an orthologous or homologous sequence of the strand.

4. The method of any preceding sentence, wherein the insert nucleotidesequence is at least 10 nucleotides long.

5. The method of any preceding sentence, wherein the insert sequencecomprises a site specific recombination site.

6. A method of nucleic acid recombination, the method comprising

-   -   (a) using nucleic acid cleavage to create first and second        breaks in a nucleic acid strand, thereby creating 5′ and 3′ cut        ends and a nucleotide sequence between the ends;    -   (b) using homologous recombination to delete the nucleotide        sequence; and    -   (c) optionally obtaining the nucleic acid strand modified in        step (b) or a progeny nucleic strand comprising the deletion.

7. The method of sentence 6, wherein the deleted sequence comprises aregulatory element or encodes all or part of a protein.

8. The method of any preceding sentence, wherein step (c) is performedby isolating a cell comprising the modified first strand, or byobtaining a non-human vertebrate in which the method has been performedor a progeny thereof.

9. The method of any preceding sentence, wherein the nucleic acid strandor the first strand is a DNA strand.

10. The method of any preceding sentence wherein the product of themethod comprises a nucleic acid strand comprising a PAM motif 3′ of theinsertion or deletion.

11. The method of any preceding sentence, wherein step (b) is performedby carrying out homologous recombination between an incoming nucleicacid comprising first and second homology arms, wherein the homologyarms are substantially homologous respectively to a sequence extending5′ from the 5′ end and a sequence extending 3′ from the 3′ end.

12. The method of sentence 11, wherein step (b) is performed by carryingout homologous recombination between an incoming nucleic acid comprisingan insert nucleotide sequence flanked by the first and second homologyarms, wherein the insert nucleotide sequence is inserted between the 5′and 3′ ends.

13. The method of sentence 12, wherein the insert is as recited in anyone of sentences 3 to 5 and there is no further sequence between thehomology arms.

14. The method of any one of sentences 11 to 13, wherein each homologyarm is at least 20 contiguous nucleotides long.

15. The method of any one of sentences 11 to 14, wherein the firstand/or second homology arm comprises a PAM motif.

16. The method of any preceding sentence, wherein Casendonuclease-mediated cleavage is used in step (a); optionally byrecognition of a GG or NGG PAM motif.

17. The method of sentence 16, wherein a nickase is used to cut in step(a).

18. The method of any preceding sentence, wherein the method is carriedout in a cell. e.g. a eukaryotic cell.

19. The method of sentence 18, wherein the method is carried out in amammalian cell.

20. The method of sentence 18, wherein the cell is a rodent (e.g.,mouse) ES cell or zygote.

21. The method of any preceding sentence, wherein the method is carriedout in a non-human mammal, e.g. a mouse or rat or rabbit.

22. The method of any preceding sentence, wherein each cleavage site isflanked by PAM motif (e.g., a NGG or NGGNG sequence, wherein N is anybase and G is a guanine).

23. The method of any preceding sentence, wherein the 3′ end is flanked3′ by a PAM motif.

24. The method of any preceding sentence, wherein step (a) is carriedout by cleavage in one single strand of dsDNA.

25. The method of any preceding sentence, wherein step (a) is carriedout by combining in a cell the nucleic acid strand, a Cas endonuclease,a crRNA and a tracrRNA (e.g., provided by one or more gRNAs) fortargeting the endonuclease to carry out the cleavage, and optionally aninsert sequence for homologous recombination with the nucleic acidstrand.

26. The method of any preceding sentence, wherein step (b) is performedby carrying out homologous recombination with an incoming nucleic acidcomprising first and second homology arms, wherein the homology arms aresubstantially homologous respectively to a sequence extending 5′ fromthe 5′ end and a sequence extending 3′ from the 3′ end, wherein thesecond homology arm comprises a PAM sequence such that homologousrecombination between the second homology arm and the sequence extending3′ from the 3′ end produces a sequence comprising a PAM motif in theproduct of the method.

27. A method of sequential endonuclease-mediated homology directedrecombination (sEHDR) comprising carrying out the method of anypreceding sentence (e.g., when according to sentence 1 using a nickaseto cut a single strand of dsDNA; or when dependent from sentence 2 or 5using a nuclease to cut both strands of dsDNA) a first time and a secondtime, wherein endonuclease-mediated cleavage is used in each step (a);wherein the product of the first time is used for endonuclease-mediatedcleavage the second time, whereby either (i) first and second nucleotidesequences are deleted the first time and the second times respectively;(ii) a first nucleotide sequence is deleted the first time and a secondnucleotide sequence is inserted the second time, (iii) a firstnucleotide sequence is inserted the first time and a second nucleotidesequence is deleted the second time; or (iv) first and second nucleotidesequences are inserted the first and second times respectively;optionally wherein the nucleic acid strand modification the second timeis within 20 or less nucleotides of the nucleic acid strand modificationthe first time.

28. The method of sentence 27, wherein the first time is carried outaccording to claim 6, wherein the incoming nucleic acid comprises nosequence between the first and second homology arms, wherein sequencebetween the 5′ and 3′ ends is deleted by homologous recombination;and/or the second time is carried out according to sentence 6, whereinstep (b) is performed by carrying out homologous recombination betweenan incoming nucleic acid comprising first and second homology arms,wherein the homology arms are substantially homologous respectively to asequence extending 5′ from the 5′ end and a sequence extending 3′ fromthe 3′ end, wherein the incoming nucleic acid comprises no sequencebetween the first and second homology arms such that sequence betweenthe 5′ and 3′ ends is deleted by homologous recombination; optionallywherein the second arm comprises a PAM motif such that the product ofthe second time comprises a PAM motif for use in a subsequent Casendonuclease-mediated method according to any one of sentences 1 to 26.

29. The method of sentence 27, wherein the first time is carried outaccording to sentence 1 or 2, wherein the incoming nucleic acidcomprises the insert sequence between the first and second homologyarms, wherein the insert sequence is inserted between the 5′ and 3′ endsby homologous recombination; and/or the second time is carried outaccording to sentence 1 or 2, wherein step (b) is performed by carryingout homologous recombination between an incoming nucleic acid comprisingfirst and second homology arms, wherein the homology arms aresubstantially homologous respectively to a sequence extending 5′ fromthe 5′ end and a sequence extending 3′ from the 3′ end, wherein theinsert sequence is inserted between the 5′ and 3′ ends by homologousrecombination; optionally wherein the second arm comprises a PAM motifsuch that the product of the second time comprises a PAM motif for usein a subsequent Cas endonuclease-mediated method according to any one ofsentences 1 to 26.

30. The method of sentence 27, wherein one of said first and secondtimes is carried out as specified in sentence 28 and the other time iscarried out as specified in sentence 29, wherein at least one sequencedeletion and at least one sequence insertion is performed.

31. The method of any preceding sentence, wherein step (a) is carriedout using Cas endonuclease-mediated cleavage and a gRNA comprising acrRNA and a tracrRNA.

32. The method of sentence 25 or 31, wherein the crRNA has the structure5′-X-Y-3′, wherein X is an RNA nucleotide sequence (optionally at least5 nucleotides long) and Y is a crRNA sequence comprising a nucleotidemotif that hybridises with a motif comprised by the tracrRNA, wherein Xis capable of hybridising with a nucleotide sequence extending 5′ fromthe desired site of the 5′ cut end.

33. The method of sentence 25, 31 or 32, wherein Y is5′-NIUUUUAN2N3GCUA-3′ (SEQ ID NO:3), wherein each of N1-3 is a A, U, Cor G and/or the tracrRNA comprises the sequence (in 5′ to 3′orientation) UAGCM1UUAAAAM2 (SEQ ID NO:4), wherein M1 is spacernucleotide sequence and M2 is a nucleotide.

34. A method of producing a cell or a transgenic non-human organism, themethod comprising

(a) carrying out the method of any preceding claim to (i) knock out atarget nucleotide sequence in the genome of a first cell and/or (ii)knock in an insert nucleotide sequence into the genome of a first cell,optionally wherein the insert sequence replaces a target sequence inwhole or in part at the endogenous location of the target sequence inthe genome; wherein the cell or a progeny thereof can develop into anon-human organism or cell; and

(b) developing the cell or progeny into a non-human organism or anon-human cell.

35. The method of sentence 34, wherein the organism or cell ishomozygous for the modification (i) and/or (ii).

36. The method of sentence 34 or 35, wherein the cell is an ES cell, iPScell, totipotent cell or pluripotent cell.

37. The method of any one of sentences 34 to 36, wherein the cell is arodent (e.g., a mouse or rat) cell.

38. The method of any one of sentences 34 to 37, wherein the targetsequence is an endogenous sequence comprising all or part of aregulatory element or encoding all or part of a protein.

39. The method of any one of sentences 34 to 38, wherein the insertsequence is a synthetic sequence; or comprises a sequence encoding allor part of a protein from a species other than the species from whichthe first cell is derived; or comprises a regulatory element from saidfirst species.

40. The method of sentence 39, wherein the insert sequence encodes allor part of a human protein or a human protein subunit or domain.

41. A cell or a non-human organism whose genome comprises a modificationcomprising a non-endogenous nucleotide sequence flanked by endogenousnucleotide sequences, wherein the cell or organism is obtainable by themethod of any one of sentences 24 to 40 and wherein the non-endogenoussequence is flanked 3′ by a Cas PAM motif; wherein the cell is notcomprised by a human; and one, more or all of (a) to (d) applies

(a) the genome is homozygous for the modification; or comprises themodification at one allele and is unmodified by Cas-mediated homologousrecombination at the other allele;

(b) the non-endogenous sequence comprises all or part of a regulatoryelement or encodes all or part of a protein;

(c) the non-endogenous sequence is at least 20 nucleotides long;

(d) the non-endogenous sequence replaces an orthologous or homologoussequence in the genome.

42. The cell or organism of sentence 41, wherein the non-endogenoussequence is a human sequence.

43. The cell or organism of sentence 41 or 42, wherein the PAM motifcomprises a sequence selected from CCN, TCN, TTC, AWG, CC, NNAGNN, NGGNGGG, NGG, WGG, CWT, CTT and GAA.

44. The cell or organism of any one of sentences 41 to 43, wherein thereis a PAM motif no more than 10 nucleotides (e.g., 3 nucleotides) 3′ ofthe non-endogenous sequence.

45. The cell or organism of any one of sentences 41 to 44, wherein thePAM motif is recognised by a Streptococcus Cas9.

46. The cell or organism of any one of sentences 41 to 45, which is anon-human vertebrate cell or a non-human vertebrate that expresses oneor more human antibody heavy chain variable domains (and optionally noheavy chain variable domains of a non-human vertebrate species).

47. The cell or organism of any one of sentences 41 to 46, which is anon-human vertebrate cell or a non-human vertebrate that expresses oneor more human antibody kappa light chain variable domains (andoptionally no kappa light chain variable domains of a non-humanvertebrate species).

48. The cell or organism of any one of sentences 41 to 47, which is anon-human vertebrate cell or a non-human vertebrate that expresses oneor more human antibody lambda light chain variable domains (andoptionally no kappa light chain variable domains of a non-humanvertebrate species).

49. The cell or organism of any one of sentences 46 to 48, wherein thenon-endogenous sequence encodes a human Fc receptor protein or subunitor domain thereof (e.g., a human FcRn or Fcγ receptor protein, subunitor domain).

50. The cell or organism of any one of sentences 41 to 48, wherein thenon-endogenous sequence comprises one or more human antibody genesegments, an antibody variable region or an antibody constant region.

51. The cell or organism of any one of sentences 41 to 50, wherein theinsert sequence is a human sequence that replaces or supplements anorthologous non-human sequence.

52. A monoclonal or polyclonal antibody prepared by immunisation of avertebrate (e.g., mouse or rat) according to any one of sentences 41 to51 with an antigen.

53. A method of isolating an antibody that binds a predeterminedantigen, the method comprising

(a) providing a vertebrate (optionally a mouse or rat) according to anyone of sentences 41 to 51;

(b) immunising said vertebrate with said antigen:

(c) removing B lymphocytes from the vertebrate and selecting one or moreB lymphocytes expressing antibodies that bind to the antigen;

(d) optionally immortalising said selected B lymphocytes or progenythereof, optionally by producing hybridomas therefrom; and

(e) isolating an antibody (e.g., and IgG-type antibody) expressed by theB lymphocytes.

54. The method of sentence 53, comprising the step of isolating fromsaid B lymphocytes nucleic acid encoding said antibody that binds saidantigen; optionally exchanging the heavy chain constant regionnucleotide sequence of the antibody with a nucleotide sequence encodinga human or humanised heavy chain constant region and optionally affinitymaturing the variable region of said antibody; and optionally insertingsaid nucleic acid into an expression vector and optionally a host.

55. The method of sentence 53 or 54, further comprising making a mutantor derivative of the antibody produced by the method of claim 53 or 54.

56. The use of an isolated, monoclonal or polyclonal antibody accordingto sentence 52, or a mutant or derivative antibody thereof that bindssaid antigen, in the manufacture of a composition for use as amedicament.

57. The use of an isolated, monoclonal or polyclonal antibody accordingto sentence 52, or a mutant or derivative antibody thereof that bindssaid antigen for use in medicine.

58. A nucleotide sequence encoding an antibody of sentence 52,optionally wherein the nucleotide sequence is part of a vector.

59. A pharmaceutical composition comprising the antibody or antibodiesof sentence 52 and a diluent, excipient or carrier.

60. An ES cell, a eukaryotic cell, a mammalian cell, a non-human animalor a non-human blastocyst comprising an expressiblegenomically-integrated nucleotide sequence encoding a Cas endonuclease.

61. The cell, animal or blastocyst of sentence 60, wherein theendonuclease sequence is constitutively expressible.

62. The cell, animal or blastocyst of sentence 60, wherein theendonuclease sequence is inducibly expressible.

63. The cell, animal or blastocyst of sentence 60, 61 or 62, wherein theendonuclease sequence is expressible in a tissue-specific orstage-specific manner in the animal or a progeny thereof, or in anon-human animal that is a progeny of the cell or blastocyst.

64. The cell or animal of sentence 63, wherein the cell is a non-humanembryo cell or the animal is a non-human embryo, wherein theendonuclease sequence is expressible or expressed in the cell or embryo.

65. The cell of animal sentence 64, wherein the endonuclease isoperatively linked to a promoter selected from the group consisting ofan embryo-specific promoter (e.g., a Nanog promoter, a Pou5fl promoteror a SoxB promoter).

66. The cell, animal or blastocyst of any one of sentences 60 to 65,wherein the Cas endonuclease is at a Rosa 26 locus.

67. The cell, animal or blastocyst of any one of sentences 60 to 65,wherein the Cas endonuclease is operably linked to a Rosa 26 promoter.

68. The cell, animal or blastocyst of any one of sentences 60 to 63,w5erein the Cas endonuclease sequence is flanked 5′ and 3′ by transposonelements (e.g., inverted piggyBac terminal elements) or site-specificrecombination sites (e.g., loxP and/or a mutant lox, e.g., lox2272 orlox511; or frt).

69. The cell, animal or blastocyst of sentence 68, comprising one ormore restriction endonuclease sites between the Cas endonucleasesequence and a transposon element.

70. The cell, animal or blastocyst of any one of sentences 60 to 69comprising one or more gRNAs.

71. The cell, animal or blastocyst of sentence 68, 69 or 70, wherein thegRNA(s) are flanked 5′ and 3′ by transposon elements (e.g., invertedpiggyBac terminal elements) or site-specific recombination sites (e.g.,loxP and/or a mutant lox, e.g., lox2272 or lox511 or frt).

72. Use of the cell, animal or blastocyst of any one of sentences 60 to71 in a method according to any one of sentences 1 to 51 or 73.

73. A method of nucleic acid recombination, the method comprisingproviding dsDNA comprising first and second strands and

(a) using Cas endonuclease-mediated nucleic acid cleavage to create acut end in the first strand 3′ of a PAM motif;

(b) using Cas endonuclease-mediated nucleic acid cleavage to create acut in the second strand at a position which corresponds to a position3′ of the cut end of the strand of part (a), which cut is 3′ of the PAMmotif:

(c) providing a first gRNA which hybridises with a sequence 5′ to thePAM motif in the strand of part (a)

(d) providing a second gRNA which hybridises with a sequence 5′ to thePAM motif in the strand of part (b) wherein the nucleic acid strands ofpart (a) and part (b) are repaired to produce a deletion of nucleic acidbetween the cuts.

74. The method of sentence 6, wherein the deleted sequence comprises aregulatory element or encodes all or part of a protein.

75. The method of sentence 73 or 74, wherein Cas endonuclease-mediatedcleavage is used in step (a) or in step (b) is by recognition of a GG orNGG PAM motif.

76. The method of sentence 75, wherein a nickase is used to cut in step(a) and/or in step (b).

77. The method of sentence 73 or 74 wherein a nuclease is used to cut instep (a) and/or in step (b).

78. The method of any one of sentences 74 to 77, wherein the method iscarried out in a cell, e.g. a eukaryotic cell.

79. The method of sentence 78, wherein the method is carried out in amammalian cell.

80. The method of sentence 78, wherein the cell is a rodent (e.g.,mouse) ES cell or zygote.

81. The method of any one of sentences 74 to 80, wherein the method iscarried out in a non-human mammal, e.g. a mouse or rat or rabbit.

82. The method of any one of sentences 74 to 81, wherein each cleavagesite is flanked by PAM motif (e.g., a NGG or NGGNG sequence, wherein Nis any base and G is a guanine).

83. Use of a first and second gRNA to target a desired part of thenucleic acid, defining the region to be deleted, in a method accordingto any one of sentences 74 to 82.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Precise DNA Insertion in a Predefined Location (KI): gRNAdesigned against a predefined location can induce DNA nick using Cas9D10A nickase 5′ of the PAM sequence (shown as solid black box).Alternatively, gRNA can be used together with Cas9 wild-type nuclease toinduce double-stranded DNA breaks 5′ of the PAM sequence. The additionof a donor oligo or a donor DNA fragment (single or double stranded)with homology around the breakpoint region containing any form of DNAalterations including addition of endogenous or exogenous DNA can beprecisely inserted at the breakpoint junction where the DNA is repairedthrough HDR.

FIG. 1B. Precise DNA Insertion in a Predefined Location (KI): Thisfigure shows a more detailed description of the mechanism described inFIG. 1A. sgRNA designed against a predefined location can induce DNAnick using Cas9 D10A nickase 5′ of the PAM sequence (shown as solidblack box). Alternatively, sgRNA can be used together with Cas9wild-type nuclease to induce double-stranded DNA breaks 5′ of the PAMsequence. The addition of a donor oligo or a donor DNA fragment (singleor double stranded) with homology arms (HA) around the breakpoint regioncontaining any form of DNA alterations including addition of endogenousor exogenous DNA, can be precisely inserted at the breakpoint junctionwhere the DNA is repaired through HDR.

FIG. 2A. Precise DNA Deletion (KO): gRNAs targeting flanking region ofinterest can induce two DNA nicks using Cas9 D10A nickase in predefinelocations containing the desired PAM sequences (shown as solid blackbox). Alternatively, gRNAs can be used with Cas9 wild-type nuclease toinduce two DSB flanking the region of interest. Addition of a donoroligo or a donor DNA fragment (single or double stranded) with homologyto region 5′ of PAM 1 and 3′ of PAM 2 sequence will guide DNA repair ina precise manner via HDR. DNA repair via HDR will reduce the risk ofindel formation at the breakpoint junctions and avoid DNA repair throughNHEJ and in doing so, it will delete out the region flanked by the PAMsequence and carry out DNA repair in a pre-determined and pre-definedmanner.

FIG. 2B. Precise DNA Deletion (KO): This figure shows a more detaileddescription of the mechanism described in FIG. 2A. sgRNAs targetingflanking region of interest can induce two DNA nicks using Cas9 D10Anickase in predefine locations containing the desired PAM sequences(shown as solid black box). Note. The PAMs can be located in oppositeDNA strands as suppose to the example depicted in the figure where bothPAMs are on the same DNA strand. Alternatively, sgRNAs can be used withCas9 wild-type nuclease to induce two DSB flanking the region ofinterest. Addition of a donor oligo or a donor DNA fragment (single ordouble stranded) with homology to region 5′ of PAM 1 and 3′ of PAM 2sequence will guide DNA repair in a precise manner via HDR. DNA repairvia HDR will reduce the risk of indel formation at the breakpointjunctions and avoid DNA repair through NHEJ and in doing so, it willdelete out the region flanked by the PAM sequence and carry out DNArepair in a pre-determined and pre-defined manner.

FIG. 3A: Precise DNA Deletion and Insertion (KO→KI): gRNAs targetingflanking region of interest can induce two DNA nicks using Cas9 D10Anickase in predefine locations containing the desired PAM sequences(shown as solid black box). Alternatively, gRNAs can be used with Cas9wild-type nuclease to induce two DSB flanking the region of interest.Addition of a donor oligo or a donor DNA fragment (single or doublestranded) with homology to region 5′ of PAM 1 and 3′ to PAM 2 withinclusion of additional endogenous or exogenous DNA, will guide DNArepair in a precise manner via HDR with the concomitant deletion of theregion flanked by DSB or nick and the insertion of DNA of interest.

FIG. 3B: Precise DNA Deletion and Insertion (KO→KI): s This figure showsa more detailed description of the mechanism described in FIG. 3A. gRNAstargeting flanking region of interest can induce two DNA nicks usingCas9 D10A nickase in predefine locations containing the desired PAMsequences (shown as solid black box). Alternatively, sgRNAs can be usedwith Cas9 wild-type nuclease to induce two DSB flanking the region ofinterest. Addition of a donor oligo or a donor DNA fragment (single ordouble stranded) with homology to region 5′ of PAM 1 and 3′ to PAM 2with inclusion of additional endogenous or exogenous DNA (DNA insert),will guide DNA repair in a precise manner via HDR with the concomitantdeletion of the region flanked by DSB or nick and the insertion of DNAof interest. Note. Once again, the PAMs can be located in opposite DNAstrands as suppose to the example depicted in the figure where both PAMsare on the same DNA strand

FIG. 4A: Recycling PAM For Sequential Genome Editing (Deletions): gRNAstargeting flanking region of interest can induce two DNA nicks usingCas9 D10A nickase in predefine locations containing the desired PAMsequences (shown as solid black box). Alternatively, gRNAs can be usedwith Cas9 wild-type nuclease to induce two DSB flanking the region ofinterest. Addition of a donor oligo or a donor DNA fragment (single ordouble stranded) with homology to region 5′ of PAM 2 and 3′ of PAM 3will guide DNA repair in a precise manner via HDR and in doing so, itwill delete out the region between PAM 2 and PAM3. This deletion willretain PAM 3 and thus acts as a site for carrying out another round ofCRISPR/Cas mediated genome editing. Another PAM site (e.g., PAM 1) canbe used in conjunction with PAM 3 sequence to carry out another round ofdeletion as described above. Using this PAM recycling approach, manyrounds of deletions can be performed in a stepwise deletion fashion,where PAM 3 is recycled after each round. This approach can be used alsofor the stepwise addition of endogenous or exogenous DNA.

FIG. 4B: Recycling PAM For Sequential Genome Editing (Deletions): Thisfigure shows a more detailed description of the mechanism described inFIG. 4B. sgRNAs targeting flanking region of interest can induce two DNAnicks using Cas9 D10A nickase in predefine locations containing thedesired PAM sequences. Alternatively, sgRNAs can be used with Cas9wild-type nuclease to induce two DSB flanking the region of interest.Addition of a donor oligo or a donor DNA fragment (single or doublestranded) with homology to region 5′ of PAM 1 (clear PAM box) and 3′ ofPAM 2 (black PAM box) will guide DNA repair in a precise manner via HDRand in doing so, it will delete out the region between PAM 1 and PAM 2.PAM sequence together with unique gRNA can be included in the intrudingDNA and targeted back into the site of editing. In this, PAM 1 sequencefor example can be recycled and thus acts as a site for carrying outanother round of CRISPR/Cas mediated genome editing. Another PAM site(eg. PAM 3, grey PAM box) can be used in conjunction with the recycledPAM 1 sequence to carry out another round of editing (i.e. Insertion) asdescribed above. Using this PAM recycling approach, many rounds ofgenome editing can be performed in a stepwise fashion, where PAM 1 isrecycled after each round. This approach can be used also for thestepwise addition of endogenous or exogenous DNA.

FIG. 5A: CRISPR/Cas mediated Lox Insertion to facilitate RMCE: gRNAstargeting flanking region of interest can induce two DNA nicks usingCas9 D10A nickase in predefine locations containing the desired PAMsequences (shown as solid black box). Alternatively, gRNAs can be usedwith Cas9 wild-type nuclease to induce two DSB flanking the region ofinterest. Addition of two donor oligos or donor DNA fragments (single ordouble stranded) with homology to regions 5′ and 3′ of each PAM sequencewhere the donor DNA contains recombinase recognition sequence (RRS) suchas loxP and lox5171 will guide DNA repair in a precise manner via HDRwith the inclusion of these RRS. The introduced RRS can be used as alanding pad for inserting any DNA of interest with high efficiency andprecisely using recombinase mediated cassette exchange (RMCE). Theretained PAM 2 site can be recycled for another round of CRISPR/Casmediated genome editing for deleting or inserting DNA of interest. Note,the inserted DNA of interest could contain selection marker such asPGK-Puro flanked by PiggyBac LTR to allow for the initial selection andupon successful integration into DNA of interest, the selection markercan be removed conveniently by expressing hyperPbase transposase.

FIG. 5B: CRISPR/Cas mediated Lox Insertion to facilitate RMCE: Thisfigure shows a more detailed description of the mechanism described inFIG. 5A. sgRNAs targeting flanking region of interest can induce two DNAnicks using Cas9 D10A nickase in predefine locations containing thedesired PAM sequences (shown as solid black box). Alternatively, sgRNAscan be used with Cas9 wild-type nuclease to induce two DSB flanking theregion of interest. Addition of two donor oligos or donor DNA fragments(single or double stranded) with homology to regions 5′ and 3′ of eachPAM sequences where the donor DNA contains recombinase recognitionsequence (RRS) such as loxP and lox5171 will guide DNA repair in aprecise manner via HDR with the inclusion of these RRS. Note. Thetargeting of the lox sites can be done sequentially or as a pool in asingle step process. The introduced RRS can be used as a landing pad forinserting any DNA of interest with high efficiency and precisely usingrecombinase mediated cassette exchange (RMCE). As detailed in FIG. 4,the PAM sequence can be recycled for another round of CRISPR/Casmediated genome editing for deleting or inserting DNA of interest. As anoption, the inserted DNA of interest could contain selection marker suchas PGK-Puro flanked by PiggyBac LTR to allow for the initial selectionand upon successful integration into DNA of interest, the selectionmarker can be removed conveniently by expressing hyperPbase transposase.

FIGS. 6A and 6B: Genome modification to produce transposon-excisableCas9 and gRNA

FIG. 6C: Single copy Cas9 Expression: A landing pad initially can betargeted into any locus of choice in mouse ES cells or any othereukaryotic cell line. The landing pad will typically contain PiggyBac 5′and 3′ LTR, selection marker such as neo for example floxed and a geneless promoter such as PGK in the general configuration shown. Targetingis done by homologous recombination and clones are selected on G418. Thenext step will involve RMCE to insert Cas9 linked via a T2A sequence toPuro-delta-tk with the option to insert single or multiple guide RNAusing the unique restriction sites (RS). The orientation of the loxsites are positioned in a manner that only once the intruding DNAcontaining the Cas9 is inserted into the landing pad, the PGK promoteron the landing pad can activate the transcription and thus theexpression of the puromycin and via the T2A transcribe and expressionCas9 production. Using this approach a single stable expression of Cas9can be achieved. Following 4-6 days of selection on puromycin, theentire Cas9 and guide RNA floxed cassette can be excised using PiggyBactransposase (Pbase) and individual clones can be analysed for genomeediting resulting from the introduced guide RNA. As an option, a stablebank cell line expressing Cas9 can be generated from which multipleengineered cell lines can be generated. To do this, onlyCas9-T2A-Puro-delta-tk will be inserted and no gRNA at the stage ofRMCE. This will produce a general single copy Cas9 expressing cell linewhere its genome can be edited by transfecting single or multiple gRNA.

FIG. 7: Schematic representing the gRNA position with respect to gene X,the structure of the targeting vector and the oligo pair used forgenotyping the resulting targeted clones.

FIG. 8: A gel image showing the genotyping results following Cas9nuclease mediated double stranded DNA break and the subsequent DNAtargeting. The genotyping shows PCR product (880 bp) specific for the5′targeted homology arm using oligo pair HAP341/HAP334. The left handgels show genotyping data from 96 ES cell clones transfected with gRNA,human Cas9 nuclease and either a circular targeting vector (plate 1) ora linear targeting vector (Plate 2). The right hand side gels shows 96ES cell clones transfected with gRNA and either a circular targetingvector (plate 3) or a linear targeting vector (Plate 4) but with nohuman Cas9 nuclease. The percentage of the clones correctly targeted isshown for each transfection.

FIG. 9: Schematic showing the position of the gRNAs on a gene to allowfor a define deletion of the region in between the two gRNA. The oligopair primer 1 and 2 was used to detect ES clones containing the specific55 bp deletion.

FIG. 10: A 3% agarose gel containing PCR products amplified from 96 ESclones transfected with gRNA 1 and 2. Primers 1 and 2 was used toamplify around the two gRNA and any clones containing the definedeletion can be seen as a smaller PCR product, which are highlighted byan asterix.

FIG. 11: PCR genotyping by amplifying the 5′ (top gel) and 3′ (bottomgel) targeted homology arms within the Rosa26 gene located on chromosome6. Correctly targeted clones yielding PCR product for both 5′ and 3′junctions are marked with an asterix.

FIG. 12: Genotyping for the correct insertion of the Cas9 DNA cassetteby PCR amplifying the 5′ (top gel) and 3′ (bottom gel) arm of theinserted DNA cassette.

FIG. 13: PCR genotyping by amplifying the region around the guide RNAand assessing the PCR product for the presence of indels. Larger indelscan be seen directly from the gel as they yielded PCR product shorterthan the expected WT DNA suggesting significant deletion. For thepositive control, genomic DNA from mouse AB2.1 was used to size thecorresponding WT PCR product. The negative control was a no DNA watercontrol.

FIG. 14: PCR amplification of the region flanking the guide RNA usingDNA extracted from pups following zygote Cas9/guide mRNA injection foranalysing indel formation. Lane 14 shows a gross deletion in that mouseand those lanes marked with an asterix indicate these mice containsmaller indels.

FIG. 15: Summary of the sequencing data from the 8 mice analysed and thedetails of the indels detected are shown. The number refers to thefrequency of that particular indel identified in the clones analysed andthe description of the indels are shown in brackets.

DETAILED DESCRIPTION OF THE INVENTION

The inventors addressed the need for improved nucleic acid modificationtechniques. An example of a technique for nucleic acid modification isthe application of the CRISPR/Cas system. This system has been shownthus far to be the most advanced genome editing system available due,inter alia, to its broad application, the relative speed at whichgenomes can be edited to create mutations and its ease of use. Theinventors, however, believed that this technology can be advanced foreven broader applications than are apparent from the state of the art.

The inventors realised that an important aspect to achieve this would beto find a way of improving the fidelity of nucleic acid modificationsbeyond that contemplated by the CRISPR/Cas methods known in the art.

Additionally, the inventors realised that only modest nucleic acidmodifications had been reported to date. It would be desirable to effectrelatively large predefined and precise DNA deletions or insertionsusing the CRISPR/Cas system.

The inventors have devised an approach for introducing one or moredesired insertions and/or deletions of known sizes into one or morepredefined locations in a nucleic acid (e.g., in a cell or organismgenome). They developed techniques to do this either in a sequentialfashion or by inserting a discrete DNA fragment of defined size into thegenome precisely in a predefined location or carrying out a discretedeletion of a defined size at a precise location. The technique is basedon the observation that DNA single-stranded breaks are preferentiallyrepaired through the HDR pathway, and this reduces the chances of indels(e.g., produced by NHEJ) in the present invention and thus is moreefficient than prior art techniques.

To this end, the invention provides:—

A method of nucleic acid recombination, the method comprising providingdouble stranded DNA (dsDNA) comprising first and second strands and

(a) using nucleic acid cleavage to create 5′ and 3′ cut ends in thefirst strand; and(b) using homologous recombination to insert a nucleotide sequencebetween the ends, thereby producing a modified first strand; therebyproducing DNA wherein the first strand has been modified by saidrecombination but the second strand has not been modified.

Optionally, the method further comprises replicating the modified firststrand to produce a progeny dsDNA wherein each strand thereof comprisesa copy of the insert nucleotide sequence. Optionally, the methodcomprises (c) isolating the progeny dsDNA, e.g., by obtaining a cellcontaining said progeny dsDNA. Replication can be effected, for examplein a cell. For example, steps (a) and (b) are carried out in a cell andthe cell is replicated, wherein the machinery of the cell replicates themodified first strand, e.g., to produce a dsDNA progeny in which eachstrand comprises the modification.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the modified DNA strand resulting from step (b) is isolated.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the method is carried out in vitro. For example, the methodis carried out in a cell or cell population in vitro.

Alternatively, optionally, in any configuration, aspect, example orembodiment of the invention, the method is carried out to modify thegenome of a virus.

Alternatively, optionally, in any configuration, aspect, example orembodiment of the invention, the method is carried out in vivo in anorganism. In an example, the organism is a non-human organism. In anexample, it is a plant or an animal or an insect or a bacterium or ayeast. For example, the method is practised on a vertebrate (e.g., ahuman patient or a non-human vertebrate (e.g., a bird, e.g., a chicken)or non-human mammal such as a mouse, a rat or a rabbit).

Optionally, in any configuration, aspect, example or embodiment of theinvention, the method is a method of cosmetic treatment of a human or anon-therapeutic, non-surgical, non-diagnostic method, e.g., practised ona human or a non-human vertebrate or mammal (e.g., a mouse or a rat).

The invention also provides:—

A method of nucleic acid recombination, the method comprising

(a) using nucleic acid cleavage to create 5′ and 3′ cut ends in a singlenucleic acid strand;(b) using homologous recombination to insert a nucleotide sequencebetween the ends, wherein the insert sequence comprises a regulatoryelement or encodes all or part of a protein; and(c) Optionally obtaining the nucleic acid strand modified in step (b) ora progeny nucleic strand comprising the inserted nucleotide sequence,e.g., by obtaining a cell containing said progeny nucleic acid strand.

In an example the progeny strand is a product of the replication of thestrand produced by step (b). The progeny strand is, for example,produced by nucleic acid replication in a cell. For example, steps (a)and (b) are carried out in a cell and the cell is replicated, whereinthe machinery of the cell replicates the modified strand produced instep (b), e.g., to produce a dsDNA progeny in which each strandcomprises the modification.

In an example, the single nucleic acid strand is a DNA or RNA strand.

In an example, the regulatory element is a promoter or enhancer.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the inserted nucleotide sequence is a plant, animal,vertebrate or mammalian sequence. e.g., a human sequence. For example,the sequence encodes a complete protein, polypeptide, peptide, domain ora plurality (e.g. one, two or more) of any one of these. In an example,the inserted sequence confers a resistance property to a cell comprisingthe modified nucleic acid produced by the method of the invention (e.g.,herbicide, viral or bacterial resistance). In an example, the insertedsequence encodes an interleukin, receptor (e.g., a cell surfacereceptor), growth factor, hormone, antibody (or variable domain orbinding site thereof), antagonist, agonist; e.g., a human version of anyof these. In an example, the inserted sequence is an exon.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the inserted nucleotide sequence replaces an orthologous orhomologous sequence of the strand (e.g., the insert is a human sequencethat replaces a plant, human or mouse sequence). For example, the methodis carried out in a mouse or mouse cell (such as an ES cell) and theinsert replaces an orthologous or homologous mouse sequence (e.g., amouse biological target protein implicated in disease). For example, themethod is carried out (e.g., in vitro) in a human cell and the insertreplaces an orthologous or homologous human sequence (e.g., a humanbiological target protein implicated in disease, e.g., a mutated form ofa sequence is replaced with a different (e.g., wild-type) humansequence, which may be useful for correcting a gene defect in the cell.In this embodiment, the cell may be a human ES or iPS or totipotent orpluripotent stem cell and may be subsequently introduced into a humanpatient in a method of gene therapy to treat and/or prevent a medicaldisease or condition in the patient).

Optionally, in any configuration, aspect, example or embodiment of theinvention, the inserted nucleotide sequence is at least 10 nucleotideslong, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,400, 500, 600, 700, 800 or 900 nucleotides, or at least 1, 2, 3, 5, 10,20, 50 or 100 kb long.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the insert sequence comprises a site specific recombinationsite, e.g., a lox, frt or rox site. For example, the site can be a loxP,lox511 or lox2272 site.

The invention also provides:—

A method of nucleic acid recombination, the method comprising

(a) using nucleic acid cleavage to create first and second breaks in anucleic acid strand, thereby creating 5′ and 3′ cut ends and anucleotide sequence between the ends;(b) using homologous recombination to delete the nucleotide sequence;and(c) optionally obtaining the nucleic acid strand modified in step (b) ora progeny nucleic strand comprising the deletion.

In an example, the progeny strand is a product of the replication of thestrand produced by step (b). The progeny strand is, for example,produced by nucleic acid replication in a cell. For example, steps (a)and (b) are carried out in a cell and the cell is replicated, whereinthe machinery of the cell replicates the modified strand produced instep (b), e.g., to produce a dsDNA progeny in which each strandcomprises the modification.

In an example, the single nucleic acid strand is a DNA or RNA strand.

In an example, the deleted sequence comprises a regulatory element orencodes all or part of a protein. In an embodiment, the deletedregulatory element is a promoter or enhancer.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the deleted nucleotide sequence is a plant, animal,vertebrate or mammalian sequence, e.g., a human sequence. For example,the sequence encodes a complete protein, polypeptide, peptide, domain ora plurality (e.g. one, two or more) of any one of these. In an example,the deleted sequence encodes an interleukin, receptor (e.g., a cellsurface receptor), growth factor, hormone, antibody (or variable domainor binding site thereof), antagonist, agonist; e.g., a non-human versionof any of these. In an example, the deleted sequence is an exon.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the deleted nucleotide sequence is replaced by an orthologousor homologous sequence of a different species or strain (e.g., a humansequence replaces an orthologous or homologous plant, human or mousesequence). For example, the method is carried out in a mouse or mousecell and the insert replaces an orthologous or homologous mouse sequence(e.g., a mouse biological target protein implicated in disease). Forexample, the method is carried out (e.g., in vitro) in a human cell andthe insert replaces an orthologous or homologous human sequence (e.g., ahuman biological target protein implicated in disease, e.g., a mutatedform of a sequence is replaced with a different (e.g., wild-type) humansequence, which may be useful for correcting a gene defect in the cell.In this embodiment, the cell may be a human ES or iPS or totipotent orpluripotent stem cell and may be subsequently introduced into a humanpatient in a method of gene therapy to treat and/or prevent a medicaldisease or condition in the patient).

Optionally, in any configuration, aspect, example or embodiment of theinvention, the deleted nucleotide sequence is at least 10 nucleotideslong, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,400, 500, 600, 700, 800 or 900 nucleotides, or at least 1, 2, 3, 5, 10,20, 50 or 100 kb long.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (c) is performed by isolating a cell comprising themodified first strand, or by obtaining a non-human vertebrate in whichthe method has been performed or a progeny thereof.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the product of the method comprises a nucleic acid strandcomprising a PAM motif 3′ of the insertion or deletion. In an example,the PAM motif is within 10, 9, 8, 7 6, 5, 4 or 3 nucleotides of theinsertion or deletion. This is useful to enable serial insertions and/ordeletions according to the method as explained further below.

Optionally, in any configuration, aspect, example or embodiment of theinvention, the product of the method comprises a nucleic acid strandcomprising a PAM motif 5′ of the insertion or deletion. In an example,the PAM motif is within 10, 9, 8, 7 6, 5, 4 or 3 nucleotides of theinsertion or deletion. This is useful to enable serial insertions and/ordeletions according to the method as explained further below.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (b) is performed by carrying out homologousrecombination between an incoming nucleic acid comprising first andsecond homology arms, wherein the homology arms are substantiallyhomologous respectively to a sequence extending 5′ from the 5′ end and asequence extending 3′ from the 3′ end. The skilled person will befamiliar with constructing vectors and DNA molecules for use inhomologous recombination, including considerations such as homology armsize and sequence and the inclusion of selection markers between thearms. For example, the incoming nucleic acid comprises first and secondhomology arms, and the insert sequence and an optional selection markersequence (e.g., neo nucleotide sequence). The arms may be at least 20,30, 40, 50, 100 or 150 nucleotides in length, for example. Wheredeletion is required, the insert is omitted (although an optionalselection marker sequence may or may not be included between the arms).

Thus, in an embodiment of the invention, step (b) is performed bycarrying out homologous recombination between an incoming nucleic acidcomprising an insert nucleotide sequence flanked by the first and secondhomology arms, wherein the insert nucleotide sequence is insertedbetween the 5′ and 3′ ends.

In another embodiment of the invention, the insert is between thehomology arms and there is no further sequence between the arms.

In an example, each homology arm is at least 20, 30, 40, 50, 100 or 150nucleotides long.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (a) is carried out using an endonuclease, e.g., anickase. Nickases cut in a single strand of dsDNA only. For example, theendonuclease is an endonuclease of a CRISPR/Cas system, e.g., a Cas9 orCys4 endonuclease (e.g., a Cas9 or Cys4 nickase). In an example, theendonuclease recognises a PAM listed in Table 1 below, for example, theendonuclease is a Cas endonuclease that recognises a PAM selected fromCCN, TCN, TTC, AWG, CC, NNAGNN, NGGNG GG, NGG, WGG, CWT, CTT and GAA. Inan example, the Cas endonuclease is a Spyogenes endonuclease, e.g., aSpyogenes Cas9 endonuclease. In an example, a S pyogenes PAM sequence orStreptococcus thermophilus LMD-9 PAM sequence is used.

In an example, the endonuclease is a Group 1 Cas endonuclease. In anexample, the endonuclease is a Group 2 Cas endonuclease. In an example,the endonuclease is a Group 3 Cas endonuclease. In an example, theendonuclease is a Group 4 Cas endonuclease. In an example, theendonuclease is a Group 7 Cas endonuclease. In an example, theendonuclease is a Group 10 Cas endonuclease.

In an example, the endonuclease recognises a CRISPR/Cas Group 1 PAM. Inan example, the endonuclease recognises a CRISPR/Cas Group 2 PAM. In anexample, the endonuclease recognises a CRISPR/Cas Group 3 PAM. In anexample, the endonuclease recognises a CRISPR/Cas Group 4 PAM. In anexample, the endonuclease recognises a CRISPR/Cas Group 7 PAM. In anexample, the endonuclease recognises a CRISPR/Cas Group 10 PAM.

In an example, Cas endonuclease-mediated cleavage is used in step (a);optionally by recognition of a GG or NGG PAM motif.

In an example, the first and/or second homology arm comprises a PAMmotif. This is useful to enable serial insertions and/or deletionsaccording to the method as explained further below.

An example of a suitable nickase is Spyogenes Cas9 D10A nickase (seeCong et al. and the Examples section below).

Optionally, in any configuration, aspect example or embodiment of theinvention, steps (a) and (b) of the method is carried out in a cell,e.g. a bacterial, yeast, eukaryotic cell, plant, animal, mammal,vertebrate, non-human animal, rodent, rat, mouse, rabbit, fish, bird orchicken cell. For example, the cell is an E coli cell or CHO or HEK293or Picchia or Saccharomyces cell. In an example, the cell is a humancell in vitro. In one embodiment, the cell is an embryonic stem cell (EScell, e.g., a human or non-human ES cell, such as a mouse ES cell) or aninduced pluripotent stem cell (iPS cell; e.g., a human, rodent, rat ormouse iPS cell) or a pluripotent or totipotent cell. Optionally, thecell is not an embryonic cell, e.g. wherein the cell is not a humanembryonic cell. Optionally, the cell is not a pluripotent or totipotentcell. In an example, the method is used to produce a human stem cell forhuman therapy (e.g., an iPS cell generated from a cell of a patient forreintroduction into the patient after the method of the invention hasbeen performed on the cell), wherein the stem cell comprises anucleotide sequence or gene sequence inserted by the method of theinvention. The features of the examples in this paragraph can becombined.

In an example, the method is carried out in a mammalian cell. Forexample, the cell is a human cell in vitro or a non-human mammaliancell. For example, a non-human (e.g., rodent, rat or mouse) zygote. Forexample, a single-cell non-human zygote.

In an example, the method is carried out in a plant or non-human mammal,e.g. a rodent, mouse or rat or rabbit, or a tissue or organ thereof(e.g., in vitro).

In an example, the 3′ or each cleavage site is flanked 3′ by PAM motif(e.g., a motif disclosed herein, such as NGG or NGGNG sequence, whereinN is any base and G is a guanine). For example, one or more or allcleavage sites are flanked 3′ by the sequence 5′-TGGTG-3′. Unlike dsDNA,the PAM is not absolutely required for ssDNA binding and cleavage: Asingle-stranded oligodeoxynucleotide containing a protospacer with orwithout a PAM sequence is bound nearly as well as dsDNA and may be usedin the invention wherein a single strand of DNA is modified. Moreover,in the presence of Mg^(2′) ions, Cas9 cuts ssDNA bound to the crRNAusing its HNH active site independently of PAM.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (a) is carried out by cleavage in one single strand ofdsDNA or in ssDNA.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (a) is carried out by combining in a cell the nucleicacid strand, a Cas endonuclease, a crRNA and a tracrRNA (e.g., providedby one or more gRNAs) for targeting the endonuclease to carry out thecleavage, and optionally an insert sequence for homologous recombinationwith the nucleic acid strand. Instead of an insert sequence, one can usean incoming sequence containing homology arms but no insert sequence, toeffect deletion as described above. In an example, the Cas endonucleaseis encoded by a nucleotide sequence that has been introduced into thecell. In an example, the gRNA is encoded by a DNA sequence that has beenintroduced into the cell.

In an example, the method is carried out in the presence of Mg²⁺.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (b) is performed by carrying out homologousrecombination with an incoming nucleic acid comprising first and secondhomology arms, wherein the homology arms are substantially homologousrespectively to a sequence extending 5′ from the 5′ end and a sequenceextending 3′ from the 3′ end, wherein the second homology arm comprisesa PAM sequence such that homologous recombination between the secondhomology arm and the sequence extending 3′ from the 3′ end produces asequence comprising a PAM motif in the product of the method. The PAMcan be any PAM sequence disclosed herein, for example. Thus, the methodproduces a modified nucleic acid strand comprising a PAM that can beused for a subsequent nucleic acid modification according to anyconfiguration, aspect, example or embodiment of the invention, wherein aCas endonuclease is used to cut the nucleic acid. This is useful, forexample, for performing sequential endonuclease-mediated homologydirected recombination (sEHDR) according to the invention, moreparticularly sCHDR described below.

Sequential Endonuclease-Mediated Homology Directed Recombination (sEHDR)The invention further provides:—

A method of sequential endonuclease-mediated homology directedrecombination (sEHDR) comprising carrying out the method of anypreceding configuration, aspect, example or embodiment of the inventiona first time and a second time, wherein endonuclease-mediated cleavageis used in each step (a); wherein the product of the first time is usedfor endonuclease-mediated cleavage the second time, whereby either (i)first and second nucleotide sequences are deleted the first time and thesecond times respectively; (ii) a first nucleotide sequence is deletedthe first time and a second nucleotide sequence is inserted the secondtime; (iii) a first nucleotide sequence is inserted the first time and asecond nucleotide sequence is deleted the second time; or (iv) first andsecond nucleotide sequences are inserted the first and second timesrespectively; optionally wherein the nucleic acid strand modificationthe second time is within 20, 10, 5, 4, 3, 2 or 1 or less nucleotides ofthe nucleic acid strand modification the first time or directly adjacentto the nucleic acid strand modification the first time.

For example, the first and second nucleotide sequences are inserted sothat they are contiguous after the insertion the second time.Alternatively, the first and second deletions are such that a contiguoussequence has been deleted after the first and second deletions have beenperformed.

In an embodiment of sEHDR, the invention uses a Cas endonuclease. Thus,there is provided:—

A method of sequential Cas-mediated homology directed recombination(sCHDR) comprising carrying out the method of any preceding claim afirst time and a second time, wherein Cas endonuclease-mediated cleavageis used in each step (a); wherein step (b) of the first time is carriedout performing homologous recombination with an incoming nucleic acidcomprising first and second homology arms, wherein the homology arms aresubstantially homologous respectively to a sequence extending 5′ fromthe 5′ end and a sequence extending 3′ from the 3′ end, wherein thesecond homology arm comprises a PAM sequence such that homologousrecombination between the second homology arm and the sequence extending3′ from the 3′ end produces a sequence comprising a PAM motif in theproduct of the method; wherein the PAM motif of the product of the firsttime is used for Cas endonuclease-mediated cleavage the second time,whereby either (i) first and second nucleotide sequences are deleted thefirst time and the second times respectively (ii) a first nucleotidesequence is deleted the first time and a second nucleotide sequence isinserted the second time; (iii) a first nucleotide sequence is insertedthe first time and a second nucleotide sequence is deleted the secondtime; or (iv) first and second nucleotide sequences are inserted thefirst and second times respectively; optionally wherein the nucleic acidstrand modification the second time is within 20, 10, 5, 4, 3, 2 or 1 orless nucleotides of the nucleic acid strand modification the first timeor directly adjacent to the nucleic acid strand modification the firsttime.

For example, the first and second nucleotide sequences are inserted sothat they are contiguous after the insertion the second time.Alternatively, the first and second deletions are such that a contiguoussequence has been deleted after the first and second deletions have beenperformed.

In an embodiment (First Embodiment), the first time is carried outaccording to the third configuration of the invention, wherein theincoming nucleic acid comprises no sequence between the first and secondhomology arms, wherein sequence between the 5′ and 3′ ends is deleted byhomologous recombination; and/or the second time is carried outaccording to the third configuration of the invention, wherein step (b)is performed by carrying out homologous recombination between anincoming nucleic acid comprising first and second homology arms, whereinthe homology arms are substantially homologous respectively to asequence extending 5′ from the 5′ end and a sequence extending 3′ fromthe 3′ end, wherein the incoming nucleic acid comprises no sequencebetween the first and second homology arms such that sequence betweenthe 5′ and 3′ ends is deleted by homologous recombination; optionallywherein the second arm comprises a PAM motif such that the product ofthe second time comprises a PAM motif for use in a subsequent Casendonuclease-mediated method according to any configuration, aspect,example or embodiment of the invention.

In an embodiment (Second Embodiment), the first time is carried outaccording to the first or second configuration of the invention, whereinthe incoming nucleic acid comprises the insert sequence between thefirst and second homology arms, wherein the insert sequence is insertedbetween the 5′ and 3′ ends by homologous recombination; and/or thesecond time is carried out according to the first or secondconfiguration of the invention, wherein step (b) is performed bycarrying out homologous recombination between an incoming nucleic acidcomprising first and second homology arms, wherein the homology arms aresubstantially homologous respectively to a sequence extending 5′ fromthe 5′ end and a sequence extending 3′ from the 3′ end, wherein theinsert sequence is inserted between the 5′ and 3′ ends by homologousrecombination; optionally wherein the second arm comprises a PAM motifsuch that the product of the second time comprises a PAM motif for usein a subsequent Cas endonuclease-mediated method according to anyconfiguration, aspect, example or embodiment of the invention.

In an example, one of said first and second times is carried out asspecified in the First Embodiment and the other time is carried out asspecified in the Second Embodiment, wherein at least one sequencedeletion and at least one sequence insertion is performed.

Optionally, in any configuration, aspect, example or embodiment of theinvention, step (a) is carried out by Cas endonuclease-mediated cleavageusing a Cas endonuclease, one or more crRNAs and a tracrRNA. Forexample, the method is carried out in a cell and the crRNA and tracrRNAis introduced into the cell as RNA molecules. For example, the method iscarried out in a zygote (e.g., a non-human zygote, e.g., a rodent, rator mouse zygote) and the crRNA and tracrRNA is injected into zygote. Inanother embodiment, the crRNA and tracrRNA are encoded by DNA within acell or organism and are transcribed inside the cell (e.g., an ES cell,e.g., a non-human ES cell, e.g., a rodent, rat or mouse ES cell) ororganism to produce the crRNA and tracrRNA. The organism is, forexample, a non-human animal or plant or bacterium or yeast or insect. Inan embodiment, the tracrRNA is in this way encoded by DNA but one ormore crRNAs are introduced as RNA nucleic acid into the cell or organismto effect the method of the invention.

Additionally or alternatively to these examples, the endonuclease may beintroduced as a protein or a vector encoding the endonuclease may beintroduced into the cell or organism to effect the method of theinvention. In another example, the endonuclease is encoded by DNA thatis genomically integrated into the cell or organism and is transcribedand translated inside the cell or organism.

In an example, the method of the invention is carried out in an ES cell(e.g., a non-human ES cell, e.g., a rodent, rat or mouse ES cell) thathas been pre-engineered to comprise an expressiblegenomically-integrated Cas endonuclease sequence (or a vector carryingthis has been include in the cell). It would be possible to introduce(or encode) a tracrRNA. By introducing a crRNA with a guiding oligosequence to target the desired area of the cell genome, one can thencarry out modifications in the cell genome as per the invention. In anexample, a gRNA as described herein is introduced into the ES cell. Thegenomically-integrated expressible Cas endonuclease sequence can, forexample, be constitutively expressed or inducibly expressible.Alternatively or additionally, the sequence may be expressible in atissue-specific manner in a progeny organism (e.g., a rodent) developedusing the ES cell.

The initial ES cell comprising a genomically-integrated expressible Casendonuclease sequence can be used, via standard techniques, to produce aprogeny non-human animal that contains the expressible Cas endonucleasesequence. Thus, the invention provides:—

A non-human animal (e.g., a vertebrate, mammal, fish or bird), animalcell, insect, insect cell, plant or plant cell comprising agenomically-integrated expressible Cas endonuclease nucleotide sequenceand optionally a tracrRNA and/or a nucleotide sequence encoding atracrRNA. The Cas endonuclease is, for example. Cas9 or Cys4. In anexample, the animal, insect or plant genome comprises a chromosomal DNAsequence flanked by site-specific recombination sites and/or transposonelements (e.g., piggyBac transposon repeat elements), wherein thesequence encodes the endonuclease and optionally one or more gRNAs. Asdescribed in the Examples below, recombinase-mediated cassette exchange(RMCE) can be used to insert such a sequence. The transposon elementscan be used to excise the sequence from the genome once the endonucleasehas been used to perform recombination. The RMCE and/ortransposon-mediated excision can be performed in a cell (e.g., an EScell) that later is used to derive a progeny animal or plant comprisingthe desired genomic modification.

The invention also provides an ES cell derived or derivable from such ananimal, wherein the ES cell comprises a genomically-integratedexpressible Cas endonuclease nucleotide sequence. In an example, the EScell is a rodent, e.g., a mouse or rat ES cell, or is a rabbit, dog,pig, cat, cow, non-human primate, fish, amphibian or bird ES cell.

The invention also provides a method of isolating an ES cell, the methodcomprising deriving an ES cell from an animal (e.g., a non-human animal,e.g., a rodent, e.g., a rat or a mouse), wherein the animal comprises agenomically-integrated expressible Cas endonuclease nucleotide sequence,as described herein.

In any of these aspects, instead of an ES cell, the cell may be an iPScell or a totipotent or pluripotent cell. Thus, an iPS or stem cell canbe derived from (e.g., a somatic cell of) a human, engineered in vitroto comprise a genomically-integrated expressible Cas endonucleasenucleotide sequence and optionally one or more DNA sequences encoding atracrRNA or gRNA. The invention, thus, also relates to such a method andto a human iPS or stem cell comprising a genomically-integratedexpressible Cas endonuclease nucleotide sequence and optionally one ormore DNA sequences encoding a tracrRNA or gRNA. This cell can be used ina method of the invention to carry out genome modification (e.g., tocorrect a genetic defect, e.g., by replacement of defective sequencewith a desired sequence, optionally with subsequent transposon-mediatedexcision of the endonuclease-encoding sequence). After optional excisionof the Cas endonuclease sequence, the iPS cell or stem cell can beintroduced into the donor human (or a different human, e.g., a geneticrelative thereof) to carry out genetic therapy or prophylaxis. In thealternative, a totipotent or pluripotent human cell is used and thensubsequently developed into human tissue or an organ or part thereof.This is useful for providing material for human therapy or prophylaxisor for producing assay materials (e.g., for implantation into modelnon-human animals) or for use in in vitro testing (e.g., of drugs).

In an example, the method uses a single guided RNA (gRNA or sgRNA)comprising a crRNA and a tracrRNA. The crRNA comprises anoligonucleotide sequence (“X” in the structure 5′-X-Y-3′ mentionedbelow) that is chosen to target a desired part of the nucleic acid orgenome to be modified. The skilled person will be able readily to selectappropriate oligo sequence(s). In an example, the sequence is from 3 to100 nucleotides long, e.g., from 3 to 50, 40, 30, 25, 20, 15 or 10nucleotides long, e.g., from or 5, 10, 15 or 20 to 100 nucleotides long,e.g., from 5, 10, 15 or 20 to 50 nucleotides long.

For example, the gRNA is a single nucleic acid comprising both the crRNAand the tracrRNA. An example of a gRNA comprises the sequence5′-[oligo]-[UUUUAGAGCUA (S NIUUUUAN2N3GCUA)]-[LINKER]-[UAGCAAGUUAAAA(SEQ ID NO:2)]-3′, wherein the LINKER comprises a plurality (e.g., 4 ormore, e.g., 4, 5 or 6) nucleotides (e.g., 5′-GAAA-3′).

For example, the crRNA has the structure 5′-X-Y-3′, wherein X is an RNAnucleotide sequence (optionally, at least 5 nucleotides long) and Y is acrRNA sequence comprising a nucleotide motif that hybridises with amotif comprised by the tracrRNA, wherein X is capable of hybridisingwith a nucleotide sequence 5′ of the desired site of the 5′ cut end,e.g., extending 5′ from the desired site of the 5′ cut.

In an example, Y is 5′-NIUUUUAN2N3GCUA-3′ (SEQ ID NO:3), wherein each ofN1-3 is a A, U, C or G and/or the tracrRNA comprises the sequence (in 5′to 3′ orientation) UAGCM1UUAAAAM2 (SEQ ID NO:4), wherein M1 is spacernucleotide sequence and M2 is a nucleotide; e.g., N1-G, N2=G and N3=A.The spacer sequence is, e.g., 5, 4, 3, 2 or 1 RNA nucleotides in length(e.g., AAG in 5′ to 3′ orientation). M2 is, for example, an A, U, C or G(e.g., M2 is a G). In an embodiment, a chimaeric gRNA is used whichcomprises a sequence 5′-X—Y-Z-3′, wherein X and Y are as defined aboveand Z is a tracrRNA comprising the sequence (in 5′ to 3′ orientation)UAGCM1UUAAAAM2 (SEQ ID NO:4), wherein M1 is spacer nucleotide sequenceand M2 is a nucleotide. In an example, Z comprises the sequence5′-UAGCAAGUUAAAA-3′ (SEQ ID NO:2), e.g., Z is5′-UAGCAAGUUAAAAUAAGGCUAGUCCG-3′ (SEQ ID NO:5). In an example, the gRNAhas the sequence:

(SEQ ID NO: 6) 5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′

When it is desired to use the present invention to insert an exogenoussequence into the nucleic acid to be modified, the exogenous sequencecan be provided on linear or circular nucleic acid (e.g., DNA).Typically, the exogenous sequence is flanked by homology arms that canundergo homologous recombination with sequences 5′ and 3′ respectivelyof the site where the exogenous sequence is to be inserted. The skilledperson is familiar with choosing homology arms for homologousrecombination.

The invention can be used in a method of producing a transgenicorganism, e.g., any organism recited herein. For example, the organismcan be a non-human organism used as an assay model to test apharmaceutical drug or to express an exogenous protein or a part thereof(e.g., a human protein target knocked-in into a non-human animal assayorganism). In another example, the invention has been used to knock-outan endogenous sequence (e.g., a target protein) in an organism, such asa non-human organism. This can be useful to assess the effect(phenotype) of the knock-out and thus to assess potential drug targetsor proteins implicated in disease. In one example, the organism is anon-human animal (e.g., a vertebrate, mammal, bird, fish, rodent, mouse,rat or rabbit) in which a human target protein has been knocked-in usingthe invention. Optionally, the invention has been used to knock out anorthologous or homologous endogenous target of the organism (e.g., anendogenous target sequence has been replaced at the endogenous positionby an orthologous or homologous human target sequence). In this way, anassay model can be produced for testing pharmaceutical drugs that actvia the human target.

In an embodiment, the organism is a non-human vertebrate that expresseshuman antibody variable regions whose genome comprises a replacement ofan endogenous target with an orthologous or homologous human sequence.In an example, the method of the invention is used to produce anAntibody-Generating Vertebrate or Assay Vertebrate as disclosed inWO2013061078, the disclosure of which, and specifically including thedisclosure of such Vertebrates, their composition, manufacture and use,is included specifically herein by reference as though herein reproducedin its entirety and for providing basis for claims herein.

In an example, an exogenous regulatory element is knocked-in using themethod. For example, it is knocked-in to replace an endogenousregulatory element.

In one aspect, the invention provides a method of producing a cell or atransgenic non-human organism (e.g., any non-human organism recitedherein), the method comprising:

(a) carrying out the method of any in any configuration, aspect, exampleor embodiment of the invention to (i) knock out a target nucleotidesequence in the genome of a first cell and/or (ii) knock in an insertnucleotide sequence into the genome of a first cell, optionally whereinthe insert sequence replaces a target sequence in whole or in part atthe endogenous location of the target sequence in the genome; whereinthe cell or a progeny thereof can develop into a non-human organism orcell; and

(b) developing the cell or progeny into a non-human organism or anon-human cell.

In an example, the organism or cell is homozygous for the modification(i) and/or (ii).

In an example, the cell is an ES cell (such as a mouse ES cell), iPScell, totipotent cell or pluripotent cell. In an example, the cell is anon-human vertebrate cell or a human cell in vitro. In an example, thecell is a plant, yeast, insect or bacterial cell.

In an example, the cell or organism is a rodent (e.g., a mouse or rat)cell or a rabbit, bird, fish, chicken, non-human primate, monkey, pig,dog, Camelid, shark, sheep, cow or cat cell.

In an example, the target sequence is an endogenous sequence comprisingall or part of a regulatory element or encoding all or part of aprotein.

In an example, the insert sequence is a synthetic sequence; or comprisesa sequence encoding all or part of a protein from a species other thanthe species from which the first cell is derived; or comprises aregulatory element from said first species. This is useful to combinegenes with new regulatory elements.

In an example, the insert sequence encodes all or part of a humanprotein or a human protein subunit or domain. For example, the insertsequence encodes a cell membrane protein, secreted protein,intracellular protein, cytokine, receptor protein (e.g., Fc receptorprotein, such as FcRn or a Fcγ receptor protein), protein of the humanimmune system or domain thereof (e.g., an Ig protein or domain, such asan antibody or TCR protein or domain, or a MHC protein), a hormone orgrowth factor.

The invention also provides:—

A cell (e.g., an isolated or purified cell, e.g., a cell in vitro, orany cell disclosed herein) or a non-human organism (e.g., any organismdisclosed herein, such as a mouse) whose genome comprises a modificationcomprising a non-endogenous nucleotide sequence flanked by endogenousnucleotide sequences, wherein the cell or organism is obtainable by themethod of any configuration, aspect, example or embodiment of theinvention, and wherein the non-endogenous sequence is flanked 3′ and/or5′ by (e.g., within 20, 10, 5, 4, 3, 2 or 1 or less nucleotides of, ordirectly adjacent to) a Cas PAM motif; wherein the cell is not comprisedby a human; and one, more or all of (a) to (d) applies (for example,(a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c);(b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c)and (d); (b), (c) and (d) or all of (a), (b), (c) and (d)).

-   (a) the genome is homozygous for the modification; or comprises the    modification at one allele and is unmodified by Cas-mediated    homologous recombination at the other allele;-   (b) the non-endogenous sequence comprises all or part of a    regulatory element or encodes all or part of a protein:-   (c) the non-endogenous sequence is at least 20, 30, 40, 50, 60, 70,    80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900    nucleotides, or at least 1, 2, 3, 5, 10, 20, 50 or 100 kb long:-   (d) the non-endogenous sequence replaces an orthologous or    homologous sequence in the genome.

The cell can be a human cell, or included in human tissue but not partof a human being. For example, the cell is a human cell in vitro.

In an example, the non-endogenous sequence is a human sequence.

In an example, the PAM motif is any PAM disclosed herein or comprises asequence selected from CCN, TCN, TTC, AWG, CC, NNAGNN, NGGNG GG, NGG,WGG, CWT, CTT and GAA. For example, the motif is a Cas9 PAM motif. Forexample, the PAM is NGG. In another example, the PAM is GG.

In an example, there is a PAM motif no more than 10 nucleotides (e.g., 3nucleotides) 3′ and/or 5′ of the non-endogenous sequence.

In an example, the PAM motif is recognised by a Streptococcus Cas9.

In an example, the cell or organism is a non-human vertebrate cell or anon-human vertebrate that expresses one or more human antibody heavychain variable domains (and optionally no heavy chain variable domainsof a non-human vertebrate species). For example, the organism is anAntibody-Generating Vertebrate or Assay Vertebrate disclosed inWO2013061078.

In an example, the cell or organism is a non-human vertebrate cell or anon-human vertebrate that expresses one or more human antibody kappalight chain variable domains (and optionally no kappa light chainvariable domains of a non-human vertebrate species).

In an example, the cell or organism is a non-human vertebrate cell or anon-human vertebrate that expresses one or more human antibody lambdalight chain variable domains (and optionally no kappa light chainvariable domains of a non-human vertebrate species).

In an example, the non-endogenous sequence encodes a human Fc receptorprotein or subunit or domain thereof (e.g., a human FcRn or Fcγ receptorprotein, subunit or domain).

In an example, the non-endogenous sequence comprises one or more humanantibody gene segments, an antibody variable region or an antibodyconstant region.

In an example, the insert sequence is a human sequence that replaces orsupplements an orthologous non-human sequence.

The invention also provides:—

A monoclonal or polyclonal antibody prepared by immunisation of avertebrate (e.g., mouse or rat) of the invention (or produced by amethod of the invention) with an antigen.

The invention also provides:—

A method of isolating an antibody that binds a predetermined antigen,the method comprising:

-   -   (a) providing a vertebrate (optionally a mouse or rat) of the        invention (or produced by a method of the invention);

-   (b) immunising said vertebrate with said antigen;

-   (c) removing B lymphocytes from the vertebrate and selecting one or    more B lymphocytes expressing antibodies that bind to the antigen:

-   (d) optionally immortalising said selected B lymphocytes or progeny    thereof, optionally by producing hybridomas therefrom; and

-   (e) isolating an antibody (e.g., an IgG-type antibody) expressed by    the B lymphocytes.

In an example, the method comprises the step of isolating from said Blymphocytes nucleic acid encoding said antibody that binds said antigen;optionally exchanging the heavy chain constant region nucleotidesequence of the antibody with a nucleotide sequence encoding a human orhumanised heavy chain constant region and optionally affinity maturingthe variable region of said antibody; and optionally inserting saidnucleic acid into an expression vector and optionally a host.

In an example, the method comprises making a mutant or derivative of theantibody produced by the method.

The invention provides the use of an isolated, monoclonal or polyclonalantibody described herein, or a mutant or derivative antibody thereofthat binds said antigen, in the manufacture of a composition for use asa medicament.

The invention provides the use of an isolated, monoclonal or polyclonalantibody described herein, or a mutant or derivative antibody thereofthat binds said antigen for use in medicine.

The invention provides a method of treating a patient in need thereof(e.g., a human patient), comprising administering a therapeuticallyeffective amount of an isolated, monoclonal or polyclonal antibodydescribed herein, or a mutant or derivative antibody thereof which bindsan antigen.

The invention provides a nucleotide sequence encoding an antibodydescribed herein, optionally wherein the nucleotide sequence is part ofa vector. The invention also provides a host cell comprising saidnucleotide sequence.

The invention provides a pharmaceutical composition comprising theantibody or antibodies described herein and a diluent, excipient orcarrier.

The invention provides an ES cell, a non-human animal or a non-humanblastocyst comprising an expressible genomically-integrated nucleotidesequence encoding a Cas endonuclease (e.g., a Cas9 or Cys4) andoptionally an expressible genomically-integrated nucleotide sequenceencoding a tracrRNA or a gRNA. For example, the ES cell is any ES celltype described herein.

In an example of the cell, animal or blastocyst, the endonucleasesequence is constitutively expressible.

In an example of the cell, animal or blastocyst, the endonucleasesequence is inducibly expressible.

In an example of the cell, animal or blastocyst the endonucleasesequence is expressible in a tissue-specific manner in the animal or aprogeny thereof, or in a non-human animal that is a progeny of the cellor blastocyst.

In an example, the cell, animal or blastocyst comprises one or moregRNAs or an expressible nucleotide sequence encoding a gRNA or aplurality of expressible nucleotide sequences each encoding a differentgRNA.

The invention provides the use of the cell, animal or blastocyst in amethod according to any configuration, aspect, embodiment or example ofthe invention.

An aspect provides an antibody produced by the method of the invention,optionally for use in medicine, e.g., for treating and/or preventing(such as in a method of treating and/or preventing) a medical conditionor disease in a patient, e.g., a human.

An aspect provides a nucleotide sequence encoding the antibody of theinvention, optionally wherein the nucleotide sequence is part of avector. Suitable vectors will be readily apparent to the skilled person,e.g., a conventional antibody expression vector comprising thenucleotide sequence together in operable linkage with one or moreexpression control elements.

An aspect provides a pharmaceutical composition comprising the antibodyof the invention and a diluent, excipient or carrier, optionally whereinthe composition is contained in an intravenous (IV) container (e.g., andTV bag) or a container connected to an IV syringe.

An aspect provides the use of the antibody of the invention in themanufacture of a medicament for the treatment and/or prophylaxis of adisease or condition in a patient, e.g. a human.

In a further aspect, the invention relates to humanised antibodies andantibody chains produced according to the present invention, both inchimaeric and fully humanised form, and use of said antibodies inmedicine. The invention also relates to a pharmaceutical compositioncomprising such an antibody and a pharmaceutically acceptable carrier orother excipient.

Antibody chains containing human sequences, such as chimaeric human-nonhuman antibody chains, are considered humanised herein by virtue of thepresence of the human protein coding regions region. Fully humanantibodies may be produced starting from DNA encoding a chimaericantibody chain of the invention using standard techniques.

Methods for the generation of both monoclonal and polyclonal antibodiesare well known in the art, and the present invention relates to bothpolyclonal and monoclonal antibodies of chimaeric or fully humanisedantibodies produced in response to antigen challenge in non-humanvertebrates of the present invention.

In a yet further aspect, chimaeric antibodies or antibody chainsgenerated in the present invention may be manipulated, suitably at theDNA level, to generate molecules with antibody-like properties orstructure, such as a human variable region from a heavy or light chainabsent a constant region, for example a domain antibody; or a humanvariable region with any constant region from either heavy or lightchain from the same or different species; or a human variable regionwith a non-naturally occurring constant region; or human variable regiontogether with any other fusion partner. The invention relates to allsuch chimaeric antibody derivatives derived from chimaeric antibodiesidentified according to the present invention.

In a further aspect, the invention relates to use of animals of thepresent invention in the analysis of the likely effects of drugs andvaccines in the context of a quasi-human antibody repertoire.

The invention also relates to a method for identification or validationof a drug or vaccine, the method comprising delivering the vaccine ordrug to a mammal of the invention and monitoring one or more of: theimmune response, the safety profile; the effect on disease.

The invention also relates to a kit comprising an antibody or antibodyderivative as disclosed herein and either instructions for use of suchantibody or a suitable laboratory reagent, such as a buffer, antibodydetection reagent.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claimsand/or the specification may mean “one,” but it is also consistent withthe meaning of “one or more,” “at least one,” and “one or more thanone.” The use of the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or.” Throughoutthis application, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”). “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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-   1. Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu    X, Jiang W, Marraffini L A et al: Multiplex genome engineering using    CRISPR/Cas systems. Science 2013, 339(6121):819-823.-   2. Wang H, Yang H, Shivalila C S, Dawlaty M M, Cheng A W, Zhang F,    Jaenisch R: One-step generation of mice carrying mutations in    multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013,    153(4):910-918.-   3. Mali P, Yang L, Esvelt K M, Aach J, Guell M, DiCarlo J E,    Norville J E, Church G M: RNA-guided human genome engineering via    Cas9. Science 2013, 339(6121):823-826.-   4. Gaj T, Gersbach C A, Barbas C F, 3rd: ZFN, TALEN, and    CRISPR/Cas-based methods for genome engineering. Trends Biotechnol    2013, 31(7):397-405.-   5. Perez-Pinera P, Ousterout D G, Gersbach C A: Advances in targeted    genome editing. Curr Opin Chem Biol 2012, 16(3-4):268-277.-   6. Shah S A, Erdmann S, Mojica F J, Garrett R A: Protospacer    recognition motifs: Mixed identities and functional diversity. RNA    Biol 2013, 10(5).-   7. Haurwitz R E, Sternberg S H, Doudna J A: Csy4 relies on an    unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J    2012, 31(12):2824-2832.-   8. Yusa K, Zhou L, Li M A, Bradley A, Craig N L: A hyperactive    piggyBac transposase for mammalian applications. Proc Natl Acad Sci    USA 2011, 108(4):1531-1536.-   9. Qiao J, Oumard A, Wegloehner W, Bode J: Novel tag-and-exchange    (RMCE) strategies generate master cell clones with predictable and    stable transgene expression properties. J Mol Biol 2009,    390(4):579-594.-   10. Oumard A, Qiao J, Jostock T, Li J, Bode J: Recommended Method    for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in    Animal Cell Biotechnology. Cytotechnology 2006, 50(1-3):93-108.

The present invention is described in more detail in the following nonlimiting exemplification.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of nucleic acid recombination, the method comprising    -   (a) using Cas endonuclease-mediated nucleic acid cleavage to        create first and second breaks in a nucleic acid strand, thereby        creating 5′ and 3′ cut ends and a nucleotide sequence between        the ends;    -   (b) using homologous recombination to delete the nucleotide        sequence; and    -   (c) optionally obtaining the nucleic acid strand modified in        step (b) or a progeny nucleic strand comprising the deletion.-   2. The method of paragraph 1, wherein the deleted sequence comprises    a regulatory element or encodes all or part of a protein.-   3. The method of paragraph 2, wherein the deleted sequence comprises    a protein subunit or domain.-   4. The method of any one of paragraphs 1 to 3, wherein the deletion    of step (b) is at least 20 nucleotides long.-   5. The method of paragraph 1, further comprising a step of inserting    a nucleotide sequence between the cut ends in (a).-   6. The method of paragraph 5, wherein the insert nucleotide sequence    comprises a PAM motif.-   7. The method of paragraph 5 or paragraph 6, wherein the insert    sequence is at least 10 nucleotides long.-   8. The method of any one of paragraphs 5 to 7, wherein recombinase    recognition sequences are used to insert the nucleotide sequence,    e.g. loxP and/or a mutant lox, e.g., lox2272 or lox511 or frt.-   9. The method of any one of paragraphs 5 to 7, wherein homologous    recombination is used to insert the insert nucleotide sequence.-   10. The method of any one of paragraphs 5 to 9, wherein the method    is carried out in a cell and the insert sequence replaces an    orthologous or homologous sequence in the cell.-   11. The method of any preceding paragraph, wherein step (c) is    performed by isolating a cell comprising the modified first strand,    or by obtaining a non-human vertebrate in which the method has been    performed or a progeny thereof.-   12. The method of any preceding paragraph, wherein the nucleic acid    strand or the first strand is a DNA strand.-   13. The method of any preceding paragraph wherein the product of the    method comprises a nucleic acid strand comprising a PAM motif 3′ of    the insertion or deletion.-   14. The method of paragraph 13, wherein the PAM motif is no more    than 10 nucleotides 3′ of the deletion.-   15. The method of any preceding paragraph, wherein step (b) is    performed by carrying out homologous recombination between an    incoming nucleic acid comprising first and second homology arms,    wherein the homology arms are substantially homologous respectively    to a sequence extending 5′ from the 5′ end and a sequence extending    3′ from the 3′ end.-   16. The method of paragraph 15, wherein step (b) is performed by    carrying out homologous recombination between an incoming nucleic    acid comprising an insert nucleotide sequence flanked by the first    and second homology arms, wherein the insert nucleotide sequence is    inserted between the 5′ and 3′ ends.-   17. The method of paragraph 15 or paragraph 16, wherein each    homology arm is at least 20 contiguous nucleotides long.-   18. The method of any one of paragraphs 15 to 17, wherein the first    and/or second homology arm comprises a recombinase recognition    sequence, such as a PAM motif.-   19. The method of any preceding paragraph, wherein Cas    endonuclease-mediated cleavage is used in step (a) and is carried    out by recognition of a GG or NGG PAM motif.-   20. The method of paragraph 19, wherein a nickase is used to cut in    step (a), and optionally, wherein the nickase is a Cas nickase.-   21. The method of any preceding paragraph, wherein the method is    carried out in a cell, e.g. a eukaryotic cell.-   22. The method of paragraph 21, wherein the method is carried out in    a mammalian cell, e.g. rodent or mouse cell, e.g. a rodent (e.g.,    mouse) ES cell or zygote.-   23. The method of any preceding paragraph, wherein the method is    carried out in a non-human mammal, e.g. a mouse or rat or rabbit.-   24. The method of any preceding paragraph, wherein each cleavage    site is flanked by PAM motif (e.g., a NGG or NGGNG sequence, wherein    N is any base and G is a guanine).-   25. The method of any preceding paragraph, wherein the 3′ end is    flanked 3′ by a PAM motif.-   26. The method of any preceding paragraph, wherein step (a) is    carried out by cleavage in one single strand of dsDNA.-   27. The method of any preceding paragraph, wherein step (a) is    carried out by combining in a cell the nucleic acid strand, a Cas    endonuclease, a crRNA and a tracrRNA (e.g., provided by one or more    gRNAs) for targeting the endonuclease to carry out the cleavage, and    optionally an insert sequence for homologous recombination with the    nucleic acid strand.-   28. The method of any preceding paragraph, wherein step (b) is    performed by carrying out homologous recombination with an incoming    nucleic acid comprising first and second homology arms, wherein the    homology arms are substantially homologous respectively to a    sequence extending 5′ from the 5′ end and a sequence extending 3′    from the 3′ end, wherein the second homology arm comprises a PAM    sequence such that homologous recombination between the second    homology arm and the sequence extending 3′ from the 3′ end produces    a sequence comprising a PAM motif in the product of the method.-   29. A method of sequential endonuclease-mediated homology directed    recombination (sEHDR) comprising carrying out the method of any    preceding paragraph a first time and a second time, wherein the    product of the first time is used for endonuclease-mediated cleavage    the second time, whereby either (i) first and second nucleotide    sequences are deleted the first time and the second times    respectively; (ii) a first nucleotide sequence is deleted the first    time and a second nucleotide sequence is inserted the second    time; (iii) a first nucleotide sequence is inserted the first time    and a second nucleotide sequence is deleted the second time; or (iv)    first and second nucleotide sequences are inserted the first and    second times respectively; optionally wherein the nucleic acid    strand modification the second time is within 20 or less nucleotides    of the nucleic acid strand modification the first time.-   30. The method of paragraph 29, wherein the first time is carried    out according to paragraph 1, wherein the incoming nucleic acid    comprises no sequence between the first and second homology arms,    wherein sequence between the 5′ and 3′ ends is deleted by homologous    recombination; and/or the second time is carried out according to    paragraph 1, wherein step (b) is performed by carrying out    homologous recombination between an incoming nucleic acid comprising    first and second homology arms, wherein the homology arms are    substantially homologous respectively to a sequence extending 5′    from the 5′ end and a sequence extending 3′ from the 3′ end, wherein    the incoming nucleic acid comprises no sequence between the first    and second homology arms such that sequence between the 5′ and 3′    ends is deleted by homologous recombination; optionally wherein the    second arm comprises a PAM motif such that the product of the second    time comprises a PAM motif for use in a subsequent Cas    endonuclease-mediated method according to any one of paragraphs 1 to    28.-   31. The method of any preceding paragraph, wherein step (a) is    carried out using Cas endonuclease-mediated cleavage and a gRNA    comprising a crRNA and a tracrRNA.-   32. The method of paragraph 27 or 31, wherein the crRNA has the    structure 5′-X-Y-3′, wherein X is an RNA nucleotide sequence    (optionally at least 5 nucleotides long) and Y is an RNA sequence    comprising a nucleotide motif that hybridises with a motif comprised    by the tracrRNA, wherein X is capable of hybridising with a    nucleotide sequence extending 5′ from the desired site of the 5′ cut    end.-   33. The method of paragraph 27, 31 or 32, wherein Y is    5′-N₁UUUUAN₂N3GCUA-3′, wherein each of N₁₋₃ is a A, U, C or G and/or    the tracrRNA comprises the sequence (in 5′ to 3′ orientation)    UAGCM₁UUAAAAM₂, wherein M₁ is spacer nucleotide sequence and M₂ is a    nucleotide.-   34. A method of nucleic acid recombination, the method comprising    providing dsDNA comprising first and second strands and    -   (a) using Cas endonuclease-mediated nucleic acid cleavage to        create a cut end in the first strand 3′ of a PAM motif;    -   (b) using Cas endonuclease-mediated nucleic acid cleavage to        create a cut in the second strand at a position which        corresponds to a position 3′ of the cut end of the strand of        part (a), which cut is 3′ of the PAM motif,    -   (c) providing a first gRNA which hybridises with a sequence 5′        to the PAM motif in the strand of part (a)    -   (d) providing a second gRNA which hybridises with a sequence 5′        to the PAM motif in the strand of part (b)        wherein the nucleic acid strands of part (a) and part (b) are        repaired to produce a deletion of nucleic acid between the cuts.-   35. A method of producing a cell or a transgenic non-human organism,    the method comprising:    -   (a) carrying out the method of any preceding paragraph to (i)        knock out a target nucleotide sequence in the genome of a first        cell and/or (ii) knock in an insert nucleotide sequence into the        genome of a first cell, optionally wherein the insert sequence        replaces a target sequence in whole or in part at the endogenous        location of the target sequence in the genome; wherein the cell        or a progeny thereof can develop into a non-human organism or        cell; and    -   (b) developing the cell or progeny into a non-human organism or        a non-human cell.-   36. The method of paragraph 35, wherein the organism or cell is    homozygous for the modification (i) and/or (ii).-   37. The method of paragraph 35 or 36, wherein the cell is an ES    cell, iPS cell, totipotent cell or pluripotent cell, optionally a    rodent (e.g., a mouse or rat) cell.-   38. The method of any one of paragraphs 35 to 37, wherein the target    sequence is an endogenous sequence comprising all or part of a    regulatory element or encoding all or part of a protein.-   39. The method of any one of paragraphs 35 to 38, wherein the insert    sequence is a synthetic sequence; or comprises a sequence encoding    all or part of a protein from a species other than the species from    which the first cell is derived; or comprises a regulatory element    from said first species.-   40. The method of paragraph 39, wherein the insert sequence encodes    all or part of a human protein or a human protein subunit or domain.-   41. A cell or a non-human organism whose genome comprises a    modification comprising a non-endogenous nucleotide sequence flanked    by endogenous nucleotide sequences, wherein the cell or organism is    obtainable by the method of any one of paragraphs 26 to 40 and    wherein the non-endogenous sequence is flanked 3′ by a Cas PAM    motif; wherein the cell is not comprised by a human; and one, more    or all of (a) to (d) applies    -   (a) the genome is homozygous for the modification; or comprises        the modification at one allele and is unmodified by Cas-mediated        homologous recombination at the other allele;    -   (b) the non-endogenous sequence comprises all or part of a        regulatory element or encodes all or part of a protein;    -   (c) the non-endogenous sequence is at least 20 nucleotides long;    -   (d) the non-endogenous sequence replaces an orthologous or        homologous sequence in the genome.-   42. The cell or organism of paragraph 41, wherein the non-endogenous    sequence is a human sequence.-   43. The cell or organism of paragraph 41 or 42, wherein the PAM    motif comprises a sequence selected from CCN, TCN, TTC, AWG, CC,    NNAGNN. NGGNG GG, NGG, WGG, CWT, CTT and GAA.-   44. The cell or organism of any one of paragraphs 41 to 43, wherein    there is a PAM motif no more than 10 nucleotides (e.g., 3    nucleotides) 3′ of the non-endogenous sequence.-   45. The cell or organism of any one of paragraphs 41 to 44, wherein    the PAM motif is recognised by a Streptococcus Cas9.-   46. The cell or organism of any one of paragraphs 41 to 45, which is    a non-human vertebrate cell or a non-human vertebrate that expresses    one or more human antibody heavy chain variable domains (and    optionally no heavy chain variable domains of a non-human vertebrate    species).-   47. The cell or organism of any one of paragraphs 41 to 46, which is    a non-human vertebrate cell or a non-human vertebrate that expresses    one or more human antibody kappa light chain variable domains (and    optionally no kappa light chain variable domains of a non-human    vertebrate species) or that expresses one or more human antibody    lambda light chain variable domains (and optionally no kappa light    chain variable domains of a non-human vertebrate species).-   48. The cell or organism of any paragraph 46 or paragraph 47,    wherein the non-endogenous sequence encodes a human Fc receptor    protein or subunit or domain thereof (e.g., a human FcRn or Fcγ    receptor protein, subunit or domain).-   49. The cell or organism of any one of paragraphs 41 to 48, wherein    the non-endogenous sequence comprises one or more human antibody    gene segments, an antibody variable region or an antibody constant    region.-   50. The cell or organism of any one of paragraphs 41 to 49, wherein    the insert sequence is a human sequence that replaces or supplements    an orthologous non-human sequence.-   51. A monoclonal or polyclonal antibody prepared by immunisation of    a vertebrate (e.g., mouse or rat) according to any one of paragraphs    41 to 50 with an antigen.-   52. A method of isolating an antibody that binds a predetermined    antigen, the method comprising    -   (a) providing a vertebrate (optionally a mouse or rat) according        to any one of paragraphs 41 to 51;    -   (b) immunising said vertebrate with said antigen;    -   (c) removing B lymphocytes from the vertebrate and selecting one        or more B lymphocytes expressing antibodies that bind to the        antigen;    -   (d) optionally immortalising said selected B lymphocytes or        progeny thereof, optionally by producing hybridomas therefrom;        and    -   (e) isolating an antibody (e.g., and IgG-type antibody)        expressed by the B lymphocytes.-   53. The method of paragraph 52, comprising the step of isolating    from said B lymphocytes nucleic acid encoding said antibody that    binds said antigen; optionally exchanging the heavy chain constant    region nucleotide sequence of the antibody with a nucleotide    sequence encoding a human or humanised heavy chain constant region    and optionally affinity maturing the variable region of said    antibody; and optionally inserting said nucleic acid into an    expression vector and optionally a host.-   54. The method of paragraph 52 or 53, further comprising making a    mutant or derivative of the antibody produced by the method of    paragraph 52 or 53.-   55. The use of an isolated, monoclonal or polyclonal antibody    according to paragraph 51, or a mutant or derivative antibody    thereof that binds said antigen, in the manufacture of a composition    for use as a medicament.-   56. The use of an isolated, monoclonal or polyclonal antibody    according to paragraph 51, or a mutant or derivative antibody    thereof that binds said antigen for use in medicine.-   57. A nucleotide sequence encoding an antibody of paragraph 51,    optionally wherein the nucleotide sequence is part of a vector.-   58. A pharmaceutical composition comprising the antibody or    antibodies of paragraph 51 and a diluent, excipient or carrier.-   59. An ES cell, a eukaryotic cell, a mammalian cell, a non-human    animal or a non-human blastocyst comprising an expressible    genomically-integrated nucleotide sequence encoding a Cas    endonuclease.-   60. The cell, animal or blastocyst of paragraph 59, wherein the    endonuclease sequence is constitutively expressible.-   61. The cell, animal or blastocyst of paragraph 59, wherein the    endonuclease sequence is inducibly expressible.-   62. The cell, animal or blastocyst of paragraph 59, 60 or 61,    wherein the endonuclease sequence is expressible in a    tissue-specific or stage-specific manner in the animal or a progeny    thereof, or in a non-human animal that is a progeny of the cell or    blastocyst.-   63. The cell or animal of paragraph 62, wherein the cell is a    non-human embryo cell or the animal is a non-human embryo, wherein    the endonuclease sequence is expressible or expressed in the cell or    embryo.-   64. The cell of animal paragraph 63, wherein the endonuclease is    operatively linked to a promoter selected from the group consisting    of an embryo-specific promoter (e.g., a Nanog promoter, a Pou5fl    promoter or a SoxB promoter).-   65. The cell, animal or blastocyst of any one of paragraphs 61 to    64, wherein the Cas endonuclease is at a Rosa 26 locus, and is    optionally operably linked to a Rosa 26 promoter.-   66. The cell, animal or blastocyst of any one of paragraphs 59 to    62, wherein the Cas endonuclease sequence is flanked 5′ and 3′ by    transposon elements (e.g., inverted piggyBac terminal elements) or    site-specific recombination sites (e.g., loxP and/or a mutant lox,    e.g., lox2272 or lox511; or frt).-   67. The cell, animal or blastocyst of paragraph 66, comprising one    or more restriction endonuclease sites between the Cas endonuclease    sequence and a transposon element.-   68. The cell, animal or blastocyst of any one of paragraphs 59 to 67    comprising one or more gRNAs.-   69. The cell, animal or blastocyst of paragraph 66, 67 or 68,    wherein the gRNA(s) are flanked 5′ and 3′ by transposon elements    (e.g., inverted piggyBac terminal elements) or site-specific    recombination sites (e.g., loxP and/or a mutant lox, e.g., lox2272    or lox511; or frt).-   70. Use of the cell, animal or blastocyst of any one of paragraphs    59 to 69 in a method according to any one of paragraphs 1 to 50.

EXAMPLES Example 1 Precise DNA Modifications (a) Use of Nickase for HDR

It has been reported that the Cas9 nuclease can be converted into anickase through the substitution of an aspartate to alanine (D10A) inthe RuvCl domain of SpCas9 (Cong et al.). It is noteworthy that DNAsingle-stranded breaks are preferentially repaired through the HDRpathway. The Cas9 D10A nickase, when in a complex with maturecrRNA:tracrRNA, can specifically induce DNA nicking at a preciselocation. With this in mind, we propose extending the application of theCRISPR/Cas system by creating a nick in a given location in a genomeusing Cas9 D10A nickase and then exploiting the HDR pathway forinserting a single-stranded DNA fragment (endogenous or exogenous) whichwill contain DNA homology (typically for recombineering, 50 bp is enoughfor efficient recombination) flanking the nicked DNA junction to bringin and insert a given DNA in a precision location; similar size homologywill be used with the present example (FIG. 1A). Guide RNA (gRNA) willbe design individually per target protospacer sequence or incorporatedinto a single CRISPR array encoding for 2 or more spacer sequencesallowing multiplex genome editing from a single CRSPR array.

(b) Example of Precise DNA Deletion

To demonstrate precise deletion using Cas9 in association with gRNA andno targeting vector or donor DNA, we designed two gRNA within a gene,which were 55 bp apart. The two gRNA were on opposite DNA strands asshown in FIG. 9.

Mouse ES cells were transfected with human Cas9 nuclease and the twogRNAs. The transfection procedure was carried out as detailed above butthe resulting clones were not selected. The transfected ES clones weregenotyped using oligos pair spanning the two gRNA (Primer 1 & 2) todetect specific 55 bp deletion (FIG. 10).

Most of the clones did not show the specific 55 bp deletion, however,clones were clearly identified which contained the defined deletion. Outof the 384 clones analysed, approximately 4% of the clones were found tocontain the specific 55 bp deletion. Note: Not all the genotyping datais shown. The clones containing the specific 55 bp deletion were furtheranalysed by sequencing the PCR products as a final confirmation (datanot shown). Furthermore, where we saw the specific deletion, we observedboth alleles to contain the specific deletion. These data confirmed thatwhen two gRNAs are used, a precise and specific deletion can be madewithout the requirement for a targeting vector. However we can assumethe efficiency of the define deletion can be greatly enhance using thetwo gRNA combination together with a targeting vector or a donor DNAfragment containing homology arms flanking the intended deletion region.

(c) Alternative Methodology for Deletion of DNA

In a separate setting, two gRNA or a single CRISPR array encodingmultiple spacer sequence can be designed flanking a gene or a region ofinterest and with the association of Cas9 D10A nickase, two separatesingle-stranded breaks can be induced. This, in association with asingle-stranded DNA fragment containing DNA homology to the 5′breakpoint junction of the first DNA nick, and DNA homology to the 3′breakpoint junction of the second nick, the region in between the twosingle stranded DNA nick can be precisely deleted (FIG. 2A).

(d) Alternative Methodology for Replacement of DNA

In an another setting, two separate gRNA or a multiplex single CRISPRarray can be designed flanking a gene or a region of interest and withthe association of Cas9 D10A nickase two separate single-stranded breakscan be induced. In this case the intruding single stranded DNA fragment(or double stranded DNA) can contain DNA sequence from either endogenousor exogenous source containing sequence for a known gene, regulatoryelement promoter etc. This single-stranded DNA fragment (or doublestranded DNA) can be brought together to replace the DNA region ofinterest flanked by DNA nick by arming it with DNA homology from the 5′region of the first nick and 3′ region from the second nick (FIG. 3A).Due to the high efficiency of the CRISPR/Cas system to cleave DNA, theabove proposed strategy will not require introduction of any selectionmarker, thus creating exact seamless genome editing in a precise anddefined manner. As an option, a selection marker can be included flankedby Piggy Bac LTRs to allow for the direct selection of correctlymodified clones. Once the correct clones have been identified, theselection marker can be removed conveniently through the expression ofhyperactive piggyBac transposase (Yusa K., Zhou L., Li M., Bradley A.,Craig N. L.: A hyperactive piggyBac transposase for mammalianapplications, Proc. Natl. Acad. Sci. USA, 2011, 108(4):1531-1536).Furthermore, the above approaches can be applied to ES cells, mammaliancells, yeast cells, bacterial cells, plant cells as well as directlyperforming in zygotes to expedite the process of homozygous genomeengineering in record time. It would be also possible to multiplex thissystem to generate multiple simultaneous DNA insertions (KI), deletions(KO) and the sequential deletion and insertion (KO→KI).

(e) Example of DNA Deletion and Insertion in a predefined location(KO→KI)

To demonstrate a desired DNA region can be manipulated using Cas9, asingle guide RNA (gRNA) was selected at a desired region (Exon 1 of geneX) FIG. 7. A targeting vector was also constructed, which containedapproximately 300 bp homology arms (5′ and 3′ HA) flanking the gRNA. Thehomology arms will hybridise exactly in the defined region and thusdelete a 50 bp region, which is intended for deletion. The targetingvector also allows for the insertion of any DNA sequence of interest. Inthis proof of concept experiment, we included an approximate 1.6 kbPGK-puromycin cassette. The guide RNA (0.5 ug) together with thetargeting vector (1 ug) and Cas9 nuclease vector (1 ug) was transfectedinto ES cells and 96 clones were picked after selection on puromycinusing the protocol described above. Note. As a test for targetingefficiency, we compared linear verses circular targeting vector. Also asa negative control, we did the same experiment using no Cas9 vector tocompare targeting efficiency via homologous recombination with andwithout Cas9 expression.

All the selected clones were puromycin resistant and the 96 clonespicked from each of the four transfections were genotyped using theoligo pair HAP341/HAP334. Correctly targeted clones yielded an 880 bpPCR product. The resulting genotyping data is shown in FIG. 8.

From the genotyping data of this experiment, it can be seen that Cas9mediated double stranded DNA break greatly improves homologousrecombination efficiency of the targeting vector as 62% and 49% of theclones using circular or linear targeting vector respectively werecorrectly targeted verse only a single targeted clone using circulartargeting vector when no Cas9 was used. Also it can be seen from thisdata that the circular targeting vector yielded slightly bettertargeting efficiency than when linear vector was used but a generalconclusion cannot be drawn from this single experiment but to say, bothcircular and linear targeting vector yielded greatly improved targetingefficiency when associated with Cas9 and a specific guide RNA. Thisexperiment also demonstrated that using Cas9 to create a define DNAbreakage can be used to delete out a defined DNA region and subsequentlyinsert any DNA fragment of interest

Example 2 Recycling PAM for Sequential Insertions or Deletions

In certain settings it may be useful to edit a genome by chromosomewalking. Using any of the three examples outlined above, it could bepossible to carry out sequential genome editing in a stepwise fashionwhereby the PAM sequence used in a previous round of CRISPR/Cas mediatedgenome editing, can be re-used to carry out multiple rounds of genomeediting such as deletions, insertions or the simultaneous deletion andinsertion. An example of sequential deletion whereby the PAM sequencefrom the previous genome editing step is recycled is shown in FIG. 4A.Using the PAM recycling approach, it is possible to carry out sequentialinsertions as well as sequential simultaneous deletion and insertion.

The PAM sequence us recycled through reintroducing it via homologousrecombination and as part of the homology arm. The PAM sequence can beoptionally accompanied by a unique guide-RNA sequence creating a novelsite within the host genome for further round of genome editing

Example 3 Rapid Insertion of Lox Sites Using CRISPR/Cas System

Targeting efficiency using conventional homologous recombination methodsin ES cells is low. In a different setting, the CRISPR/Cas system can beused to rapidly and efficiently introduce lox sites or other recombinaserecognition sequence such as Frt in a defined location to act as alanding pad for genome editing using recombinase mediated cassetteexchange (RMCE) (Qiao J., Oumard A., Wegloehner W., Bode J.: Noveltag-and-exchange (RMCE) strategies generate master cell clones withpredictable and stable transgene expression properties, J. Mol. Biol.,2009, 390(4):579-594; and Oumard A., Qiao J., Jostock T., Li J., BodeJ.: Recommended Method for Chromosome Exploitation: RMCE-basedCassette-exchange Systems in Animal Cell Biotechnology, Cytotechnology,2006, 50(1-3):93-108). Once the lox sites are introduced into thegenome, inversion, deletion or cassette exchange to delete and introduceDNA fragment varying in size at this site can be efficiently conductedvia expression of Cre recombinase. An example of CRISPR/Cas mediated loxinsertion followed by RMCE is shown in FIG. 5A. The RMCE step can beused to invert the region flanked by lox site or to delete this regionas well as to simultaneously delete and insert DNA of interest in thisregion. Furthermore, the RMCE step can be adapted for carrying outmultiple sequential rounds of RMCE (sRMCE).

Example 4A

Reference is made to FIG. 6A. A piggyBac transposon harbouring a PGKpromoter-driven loxP/mutant lox-flanked neo^(R) gene is targeted into anES cell genome by standard homologous recombination. The targeted clonescan be selected by G418. This provides a landing pad for the followingrecombinase-mediated cassette exchange (RMCE). Such an ES clone can beused a parental cells for any modification further. A cassettecontaining the loxP/mutant lox-flanked promoterless PuroΔTK-T2A-Cas9 andU6 polymerase III promoter-driven guide RNA (gRNA) genes are insertedinto the landing pad through transient cre expression. The gRNA genescan be one or more than one which target to the same gene or differentgenes. The inserted clones can be selected with puromycin and confirmedby junction PCRs. During the selection, the expression of Cas9 and gRNAsfrom the inserted cassette results in more efficient gene targeting ormodification than transient expression of the Cas9 and gRNA can achieve.Following 4-6 day selection, the whole modified cassette is excised bythe transient expression of piggyBac transposase (PBase). The final EScell clones would not contain any Cas9 or gRNA sequence. The clones withhomozygous modified genes would be confirmed by PCR and sequence.

The main feature of this invention is to control the Cas9 and gRNAexpression in certain time to be sufficient to generate efficienttargeting rates.

Example 4B Single Copy Cas9 Expression

As detailed in example 6, to demonstrate the single and stableexpression of Cas9 from within the chromosome of a cell, we targeted alanding pad vector into Rosa26 allele on chromosome 6. DNA homology armswere used to target the landing pad vector in between exons 2 and 3 ofRosa26. The landing pad vector was targeted into ES cells usingprocedure described above. The transfected ES clones were selected onG418 and genotyped for correct targeting (FIG. 11) by PCR amplifying the5′ and 3′ homology arm junctions.

Targeting of the landing pad yielded many targeted ES clones. Aselection of the targeted clones were used to insert a DNA cassettecontaining Cas9 nuclease linked to Puro-delta-tk via a T2A sequence intothe targeted landing pad via RMCE, which involved the expression of Crerecombinase. The corresponding loxP and lo2272 sites within both thelanding pad and the incoming vector ensured correct orientation ofinsertion. Since the landing pad contained a geneless PGK promoter,correct insertion of the incoming vector DNA containing Cas9, activatedexpression of puromycin and thus clones were positively selected onpuromycin. Non-specific targeting of this DNA cassette will not yieldpuromycin resistant clones due to the absence of a promoter driving thetranscription of the promoterless puromycin gene in the inserted DNAcassette. The initial Cas9 vector inserted into the landing pad did notcontain any guide RNA sequence. The puromycin resistant ES clones weregenotyped by PCR for the correct insertion of Cas9 (FIG. 12).

As expected owing to the positive selection, most of the clonesgenotyped for insertion of the Cas9 vector were correctly targeted viaRMCE based on the PCR genotyping results. Two of the correct clones(KHK1.6 Z2-24-27 and KHK1.10Z2-25-4 referred to as positive Z clones)which now contain the single copy Cas9 integrated into the Rosa26 geneas a single copy were used to test whether the Cas9 expression wassufficient enough to induce Cas9 mediated genome editing. Into the twopositive Z clones, guide RNA against a gene referred to as gene Y wastransfected using procedure described above. Following transfection andexpansion of the resulting ES clones, 36 individual clones were isolatedfrom each transfection and analysed initially by PCR using oligoflanking the guide RNA (FIG. 13).

Most of the clones yielded a PCR product of size equivalent to thepositive control PCR where DNA from mouse AB2.1 ES cells was used.However, it can be seen clearly that some clones yielded a PCR productdistinctively smaller than that of the positive control suggesting theseclones contain a significant deletion via indel. To verify this and tocheck whether the rest of the PCR products though similar in size to thepositive control did not contain indels, all the PCR products werepurified using Qiagen gel extraction kit and analysed by sequencing. Thesequencing data confirmed significant deletion for those PCR productsthat yielded shorter products than the positive control. It alsohighlighted, some of the other clones with similar PCR product size tothe positive control to contain indels, which included variouscombinations of insertion and deletion (Sequencing data not shown). Outof the clones analysed, 18% of them contained an indel. These dataclearly demonstrated that a single copy expression of Cas9 can be usedto carry out genome editing and these clones can now be used as a Cas9host cells for carrying out a multitude of genome editing. These ESclones are now being used to generate transgenic mouse lines whereby wecan carry out a one-step genome editing by injecting only guide mRNAdirectly into zygotes without the requirement for transcribing Cas9 mRNAto simplify the one-step genome editing protocol.

Example 5(A) Methodology A: Reconstructing CRISPR/Cas Vector System(Nuclease)

The CRISPR/Cas genome editing system has been reconstructed in vitro andexemplified in mouse embryonic stem cells using vector pX330 containinghumanised S. pyogenes (hSpCsn1) (Cong et al.). The CRISPR/Cas system canbe reconstructed as described m Cong et al using synthetic DNA stringsand DNA assembly. In the present example, the entire DNA assembly wouldconstitute a 6006 bp fragment containing 45 bp homology to pBlueScriptKS+ vector 5′ to the EcoRV cutting site. Human U6 promoter, two BbsIrestriction sites for cloning in the spacer sequence which fuses to achimeric guided RNA sequence, chicken beta-actin promoter with 3 FLAG,nuclear localisation signal (NLS) followed by hSpCsn1 sequence andanother NLS, bGH polyA, inverted terminal repeat sequence and finallyanother 45 bp homology to pBlueScript KS+3′ to the EcoRV cutting site.This 6006 bp stretch of DNA will be synthesized as 7 individual DNAfragments where each fragment will have a 45 bp overlap to the adjacentDNA fragment to allow DNA assembly. The DNA sequence of these fragmentsis shown below in the order of assembly.

Fragment 1A (1340 bp) (SEQ ID NO: 7)GGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATGAGGGCCTATTTCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGACCGGTGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTFragment 2 (852 bp) (SEQ ID NO: 8)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCFragment 3 (920 bp) (SEQ ID NO: 9)GGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCC Fragment 4 (920 bp)(SEQ ID NO: 10)CGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG Fragment 5 (920 bp)(SEQ ID NO: 11)ACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATC Fragment 6 (789 bp)(SEQ ID NO: 12)AGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACFragment 7 (535 bp) (SEQ ID NO: 13)GGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGAATTCCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCC

To reconstruct the CRISPR/Cas system described in Cong et al the aboveDNA fragments in addition to EcoRV linearised pBlueScript KS+ vectorwill be assembled using Gibson Assembly kit (NEB Cat No. E5510S). As analternative approach, the 6006 bp fragment can be assembled by assemblyPCR by mixing molar ratio of the individual DNA fragments together andusing the DNA mixture as PCR template. The assembled PCR product canthen be cloned directly into pBlueScript vector or a standard cloningvector system such as a TOPO TA cloning kit (Invitrogen).

B: Reconstructing CRISPR/Cas Vector System (D10A Nickase)

The D10A nickase version of the CRISPR/Cas system can be convenientlyreconstructed by assembling the above fragments where fragment 2 isreplaced with fragment 2A which contains the D10A substitution (Seesequence below).

Fragment 2A (852 bp) (SEQ ID NO: 14)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTG gcc ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGA GC

The substituted aspartate to alanine is highlighted in bold andunderlined.

C: Target (Spacer) Sequence Cloning

The target spacer sequence can be cloned into the above CRISPR/Casvector system via the BbsI restriction sites located upstream of thechimeric guided RNA sequence. The spacer sequence can be ordered asoligo pairs and annealed together with overhangs as shown below to allowdirect cloning into BbsI linearised CRISPR/Cas vector using standardmolecular biology protocols.

Sequence of an example oligo pair with spacer sequence:

(SEQ ID NO: 15) 5′-CACCGNNNNNNNNNNNNNNNNNNN-3′ (SEQ ID NO: 16)3′-CNNNNNNNNNNNNNNNNNNNCAAA-5′

The 4 bp overhang sequence underlined is required to be included in thespacer oligos to facilitate cloning into the BbsI restriction site inthe CRISPR/Cas vector. Using this approach, any spacer sequence can beconveniently cloned into the CRISPR/Cas vector.

D: Reconstructing CRISPR/Cas System for One-Step Generation ofTransgenic Animals

In order to reconstitute a CRISPR/Cas system for one-step generation oftransgenic animal as described in Wang et al. (Wang H., Yang H.,Shivalila C. S., Dawlaty M. M., Cheng A. W., Zhang F., Jaenisch R.:One-step generation of mice carrying mutations in multiple genes byCRISPR/Cas-mediated genome engineering, Cell, 2013, 153(4):910-918)where direct embryo injection is used, the above detailed CRISPR/Casvector system needs to be modified to incorporate a T7 polymerasepromoter to the Cas9 coding sequence. In addition, the gRNA needs to beremoved and synthetised separately by annealing oligos or producedsynthetically (See below for an example T7-spacer sequence fused tochimeric guided RNA sequence—T7-gRNA). Note, ideally the spacer sequencewill be designed in a unique region of a given chromosome to minimiseoff-target effect and also the respective protospacer genomic sequenceneeds to have a PAM at the 3′-end.

Example T7 gRNA Sequence

(SEQ ID NO: 17) TTAATACGACTCACTATAGG NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

The underlined 20 bp of N's depicts the spacer sequence for a giventarget DNA.

To reconstruct the one-step CRISPR/Cas system, the above detailed DNAfragments (Fragments 2, 3, 4, 5, 6 & 7) can be assembled together wherefragment 1A (containing 45 bp homology to pBlueScript KS+ vector 5′ tothe EcoRV restriction site, human U6 promoter, BbsI restriction sites,chimeric guided RNA sequence and chicken b-actin promoter) is replacedwith fragment 1, which only contains 45 bp homology to pBlueScript KS+vector and the DNA sequence for T7 polymerase promoter with 45 bphomology to fragment 2. This will create the nuclease version of theCRISPR/Cas system for one-step generation of transgenic animals. Tocreate the nickase version, fragment 2 can be replaced with fragment 2Aas detailed above and then fragments 1, 2A, 3, 4, 5, 6 and 7 can beassembled together either by Gibson assembly or by assembly PCR.

Fragment 1 (111 bp) (SEQ ID NO: 18)GGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGACGGAGACTACAAG GATCATGATATT

E: Preparation of Oligo/DNA Fragments for HDR-Mediated Repair

DNA oligos ranging from 15 bp and upwards in excess of >125 bp will besynthetised through Sigma Custom Oligo synthesis Service. The oligos cancontain any sequence such as a defined mutation, introduced restrictionsites or a sequence of interest including recombination recognitionsequence such as loxP or derivatives thereof, Frt and derivativesthereof or PiggyBac LTR or any other transposon elements or regulatoryelements including enhancers, promoter sequence, reporter gene,selection markets and tags. The oligo design will incorporate DNAhomology to the region where Cas9 mediates double-stranded DNA break orDNA nick. The size of the homology will range from a few base pairs (2-5bp) to upwards and in excess of 80 bp. Larger DNA fragments (>100 bpranging up to several kilobases) will be prepared either synthetically(GeneArt) or by PCR. The DNA fragment will be synthetised either with orwithout flanked NLS or only with a single NLS and either with or withouta promoter (e.g. T7 polymerase promoter). The DNA can be prepared as asingle stranded DNA fragment using either the capture biotinylatedtarget DNA sequence method (Invitrogen: Dynabeads M-270 Streptavidin) orany other standard and established single stranded DNA preparationmethodology. The single stranded DNA can be prepared for microinjectionby IVT as described above and the mRNA co-injected with Cas9 mRNA andgRNA. The DNA fragment can also be co-injected as a double stranded DNAfragment. The DNA fragment will be flanked by DNA homology to the sitewhere Cas9 mediates double-stranded DNA break or DNA nick. The DNAhomology can range from a few base pairs (2-5 bp) and up to or in excessof several kilobases. The DNA fragment can be used to introduce anyendogenous or exogenous DNA.

HDR-mediated repair can also be done in ES cells followingCRISPR/Cas-mediated DNA cleavage. The above mentioned donor oligo or DNAfragment can be co-transfected into ES cells along with the CRISPR/Casexpression vector.

F: Production of Cas9 mRNA and gRNA

Vector containing the T7 polymerase promoter with the coding region ofhumanised Cas9 will be PCR amplified using oligos Cas9-F and Cas9-R. TheT7-Cas9 PCR product can be gel extracted and the DNA purified usingQiagen gel extraction kit. The purified T7-Cas9 DNA will be used for invitro transcription (IVT) using mMESSAGE mMACHINE T7 Ultra Kit (LifeTechnologies Cat No. AM1345). The vector containing the T7-gRNA can bePCR amplified using oligos gRNA-F and gRNA-R and once again the PCRproducts gel purified. IVT of the T7-gRNA will be carried out usingMEGAshortscript T7 Kit (Life Technologies Cat No. AM1354) and the gRNApurified using MEGAclear Kit (Life Technologies Cat No. AM1908) andeluted in RNase-free water.

(SEQ ID NO: 19) Cas9-17: TTAATACGACTCACTATAGG (SEQ ID NO: 20) Cas9-R:GCGAGCTCTAGGAATTCTTAC (SEQ ID NO: 21) gRNA-F: TTAATACGACTCACTATAGG(SEQ ID NO: 22) gRNA-R: AAAAAAGCACCGACTCGGTGCCAC

Example 5B One Step Generation of Transgenic Animals A: ES CellTransfection Procedure

Mouse embryonic stem cells AB2.1 and derivatives of this line will beused for transfecting the mammalian codon optimised Cas9 and sgRNA froma single expression vector or from separate vectors if desired. AB2.1 EScells will be cultured on a PSNL76/7/4 MEF feeder layer in M-15:Knockout DMEM (Gibco, no pyruvate, high glucose, 15% FBS, 1×GPS, 1×BME)with standard ES cell culturing techniques. Transfection of CRISPR/Casexpression vector along with the optional addition of a donor oligo orDNA fragment will be done by electroporation using the Amaxa4D-Nucleofector® Protocol (Lonza). A plasmid expressing PGK-Puro willalso be optionally co-transfected to promote transfection efficiency.

In one method, after transfection ES cells will be plated back ontofeeder plates and Puromycin (2 μg/ml) will be added 72 hours posttransfection for 7 days after which colonies will be picked andgenotyped by PCR. Positive colonies will be further cultured andexpanded on feeder layer and selection markers where necessary will beexcised using a PiggyBac transposon system. This will be done byelectroporation of ES cells with a plasmid containing HyPbase using theAmaxa 4D-Nucleofector® Protocol (Lonza). The ES cell will be plated backonto feeder plates. ES cells will be passaged 2-3 days post transfectionand after a further 2-3 days the ES cells will be plated out atdifferent cells densities (1:10, 1:20, 1:100 and 1:300) and FIAU (2μg/ml) selection will be added 24 hours after replating. Colonies willbe picked and analysed by PCR genotyping after 7-10 days on selectionmedia. Positive clones will be further cultured and expanded on feederlayer and sent for zygote (blastocyst) microinjection.

In an alternative method, 8 hours prior to transfection ES cells areseeded at a density of 0.5×106 cells using antibiotic free M-15 KnockoutDMEM (Gibco, no pyruvate, high glucose, 15% FBS, 1×L-Glutamine, 1×BME)onto 6 w feeder plates. Transient transfection is performed usingLipofectamine LTX Reagent with PLUS™ Reagent (Invitrogen™) by standardprotocol. After incubation time transfection reagents are transferred tofeeder plates (cultured in antibiotic free media), media (M-15) will notbe changed on these plates for at least 24 hours post transfection. 48hours post transfection ES cells are trypsinized into a single cellsuspension and a cell count is carried out and cells are plated out atdifferent cell densities ranging for 100-5000 cells per 10 cm feederplate. 24 hours after replating Puro selection at 2 μg/ml (Puromycindihydrochloride from Streptomyces alboniger powder, P8833 Sigma) isapplied to the cells for 4 days, after which cells are cultured again inM-15. Colonies are picked 10-13 days post transfection.

Method 5C: Microinjection of Mouse Zygotes—Method 1 Materials andReagents:

-   -   M2 (Sigma M7167)    -   Embryo Max KSOM (Speciality media MR-020P-F)    -   Hyaluronidase (Sigma H4272)    -   Mineral Oil (Sigma, M-8410)

Possible Donor Strains:

-   -   S3F/S3F;KF3/KF3    -   S3F/S3F;K4/K4    -   S7F/S7F    -   K5F/K5F

Preparation of Zygotes and Microinjection:

The protocol is as described in: A. Nagy Et al. Manipulating the MouseEmbryo 3rd Edition. Chapter 7, Protocols 7-1, 7-6, 7-10, 7-11. ColdSpring Harbor Laboratory Press.

In brief:

-   1. Zygotes are harvested from E0.5 dpc (day post-coitum)    superovulated female mice.-   2. The zygotes are incubated in hyaluronidase to disperse cumulus    cells.-   3. Zygotes are collected and transferred to several drops of M2    medium to rinse off the hyaluronidase solution and debris. Zygotes    are placed into KSOM Media and incubated at 37° C., 5% CO₂ until    required.-   4. Zygote quality is assessed and zygotes with normal morphology are    selected for injection, these are placed in KSOM media and incubated    at 37° C., 5% CO₂ until required.

Microinjection Set Up:

Injection procedures are performed on a Nikon Eclipse Ti invertedmicroscope with Eppendorf micromanipulators and an Eppendorf femtojetinjection system. A slide is prepared by adding a large drop ˜200microlitres of M2 into the centre.

Microinjection:

Place an appropriate number of zygotes onto the slide. Examine thezygotes and select only those with normal morphology (2 distinctpronuclei are visible). Whilst holding a zygote with a male pronucleusclosest to the injection pipette, carefully push the injection pipettethrough the zona pellucida into the pronucleus, apply injectionpressure, the pronucleus should visibly swell, remove the injectionpipette quickly. The injected zygote can be placed down while the restare injected.

At the end of the injection session all viable injected zygotes shouldbe placed into prepared dishes containing drops of KSOM and incubateduntil ready to surgically implant. They are incubated for 2-3 hoursbefore surgically implanting into pseudo pregnant females. Pups will beborn 21 days later.

Method 5C: Microinjection of Mouse Zygotes—Method 2 Materials andReagents

-   -   PMSG    -   hCG    -   M2 (Sigma M7167)    -   Embryo Max KSOM (Specialty media MR-020P-F)    -   Mineral Oil (Sigma, M-8410)    -   Hyluronidase (Sigma H 4272)    -   35 mm Falcon Petri dishes (Fisher 08-757-100A)    -   Sharp scissors    -   Sharp watchmakers forceps

Preparation of Oocytes:

-   -   1. Day 0: Give PMSG (5 I.U.) to the females by I. P, injection.    -   2. Day 2: Give hCG (5 I.U.) to the females 48 Hours later by I.        P, injection. Mate the females to stud males.    -   3. Day 3: Check plugs, sacrifice plugged female mice by CO2        asphyxiation or cervical dislocation at 0.5 dpc at 8.00 am.    -   4. Dissect open the abdomen, locate the ovary and fat pad,        dissect out the oviduct leaving the ovary and fat, trimming the        uterine horn to ˜1 cm, place into a 35 mm Petri dish containing        M2 at room temp.    -   5. Place one ovary at a time into a dish containing        hyaluronidase solution in M2 (˜0.3 mg/ml) at room temp. View        through a stereoscope at 20× or 40× magnification.    -   6. Use one pair of forceps to grasp the oviduct and hold it on        the bottom of the dish. Use the second pair of forceps or a 26 g        needle to tear the oviduct close to where the zygotes are        located (the ampulla), releasing the clutch of cumulus cells.    -   7. The zygotes should be left in the hyaluronidase for a few        minutes only, after which time the zygotes may become damaged.        If necessary pipette them up and down a few times to help the        release of the zygotes from the cumulus cells.    -   8. Use a mouth pipette to pick up the zygotes and transfer them        to a fresh dish of M2, then transfer through several drops of M2        to rinse off the hyaluronidase, cumulus cells and debris. Sort        through the zygotes removing any obviously bad ones (fragmented,        misshapen, not fertilized), and place the good ones (two polar        bodies should be visible and any with polar bodies) into        equilibrated drops of KSOM+AA at 37° C. and 5% CO₂, keep        incubated until needed. Place about 50 eggs per drop.

Pronuclear Microinjection

-   -   1. Microinjection set up: Injection procedures are performed on        a Nikon Eclipse Ti inverted microscope with Eppendorf        micromanipulators. Prepare a 60 mm petri dish to place injected        zygotes into. Pipette four-six 40 μl drops of KSOM+AA, cover        with oil and place in a 5% CO₂ incubator to equilibrate. Prepare        a cavity slide by making a large (˜200 μl) drop of M2 media onto        the center of the well, add a small drop of medium on the left        side of the slide, for the holding pipette.    -   2. Microinjection: Ensure that the pressurized injector has been        switched on and is ready to use. Place an appropriate number of        zygotes onto the slide, do not add more zygotes than can be        injected within 20-30 mins. Place the holding pipette into the        drop of M2 on the left of the slide; it will fill using        capillary action, once filled to about the shoulder attach to        the manipulator. Carefully examine the zygotes, making sure that        two pronuclei are visible and morphology is good, discard any        that appear abnormal. To test if the injection needle is open,        place the tip near to but not touching a zygote in the same        focal plane. Apply pressure using the pressurized system, if the        zygote moves the needle is open, if it doesn't the needle is        closed. In this case apply pressure using the “clear” feature,        if the tip is still not open manually break the tip. Carefully        “knock” the tip on the holding pipette and repeat the above        test, make sure the tip does not become too large, if this        happens replace the needle and start again. Place the tip of the        holding pipette next to a zygote and suck it onto the end of the        pipette by applying negative pressure. Focus the microscope to        locate the pronuclei, the zygote should be positioned in such a        way that allows injection into the zygote without hitting the        pronuclei, preferably with a gap between the zona pellucida and        the oolema. Bring the tip of the injection needle into the same        focal plane as the zona pellucida. Bring the injection pipette        to the same y-axis position as the zona pellucida, adjust the        height of the needle so the tip appears completely sharp,        without changing the focus. This ensures the needle will target        the zygote exactly. Push the injection pipette through the zona        pellucida, through the cytoplasm towards the back of the zygote.        The needle will create a “bubble” through the oolema, this needs        to be broken, you will see it snap back at which point remove        the needle quickly, you will see the cytoplasm moving to        indicate RNA is flowing from the needle. Cytoplasmic granules        flowing out of the oocytes after removal of the injection        pipette is a clear sign that the zygote will soon lyse. In this        case, or if nuclear/cytoplasmic components are sticking to the        injection pipette, the oocytes should be discarded after        injection. If the zygote appears to be intact and successfully        injected, sort this into a good group. Pick a new zygote for        injection. The same injection pipette can be used as long as it        continues to inject successfully. Switch to a new injection        pipette if (a) you cannot see any cytoplasmic distortion (b)        zygotes are lysing one after the other, (c) the tip of the        pipette becomes visibly “dirty” or nuclear contents stick to the        pipette. Once all the zygotes have been injected, remove them        and place them into the equilibrated KSOM+AA and place them into        the incubator at 37° C. overnight. Only transfer those zygotes        that have survived injection, and cultured to the 2 cell stage.        Leave any lysed ones, and zygotes that have not developed.    -   3. Count the total number injected and record the numbers        transferred per recipient

Results

To demonstrate the efficient of the one-step generation of transgenicmice, we used our T7-Cas9 nuclease vector to generate mRNA via in vitrotranscription detailed above. mRNA from the guide RNA was also producedusing in vitro transcription described above. Before injecting the mRNAmixture into the cytoplasm, oocytes were prepared from female mice usingthe protocol detailed above. An mRNA mixture containing 100 ng/ul Cas9nuclease mRNA and 50 ng/ul guide mRNA was injected by microinjectioninto the cytoplasm as detailed above. The microinjection is done at thesingle-cell stage. Zygotes that survived the injection were cultured to2 cell stage, which were then transferred to recipient mice.

In total, 107 zygotes were injected from which 49 survived and went to 2cell stage. These were then transferred to two recipient female mice.This resulted in 19 pups from 2 litters. Litter 1 yielded 3 males and 6females. Litter 2 yielded 4 males and 6 females. The pups were earclipped 3 weeks after birth and DNA was extracted. PCR was carried outusing oligos flanking the gRNA to detect possible indels (FIG. 14).

PCR amplifying around the guide RNA and separating out the PCR productson an agarose gel highlighted at least one mouse contained a large indelin the form of a deletion, whereas other mice appeared to have smallerindels judging by the sharpness of the PCR product on the gel. As aninitial crude analysis, all the PCR products were sent for sequencingand those marked with an asterix (7 mice in total. FIG. 14) yielded mixsequences around the gRNA further confirming they contain indels. Toconfirm this, the PCR products from these 7 mice together with the PCRproduct from another mouse which did not yield a mix sequence (PCRproduct from lane 19, FIG. 14) were individually cloned into a generalcloning vector. From each individual cloning, 28 clones were picked andanalysed by sequencing. The sequencing confirmed all 7 mice containindels and the mice that did not contain any mix sequence contained noindels. The sequencing data is summarised in FIG. 15.

The sequencing data confirmed all of the mice analysed contained indels.It also suggests that using our zygote injection protocol detailed aboveand our method for preparing mRNA for Cas9 and guide RNA, Cas9 worksefficiently at an early stage and until the point where cells starts todivide beyond the 2 cell stage judging by the fact that in all of themice analysed, no more than 3 types of indels were identified. Out ofthe 7 mice containing indels, 3 of them had no detectable WT sequence.The female mouse (KMKY6.1j) that did not show mix sequence from theinitial sequencing analysis indeed did not contain any indels so itvalidates our initial sequencing analysis of the PCR products.

The male mouse (KMKY5.1c) that showed no WT sequence was used as amating partner for the two female mice (KMKY5.1e & KMKY6.1e) that showedno WT sequence too. The resulting pups from the two matings yielded 14pups in total from the first litter. Following similar sequencinganalysis whereby PCR products amplified from the region around the guideRNA were cloned individual and several clones were then analysed for thepresence of indels. For each mouse, 24 clones were analysed bysequencing. The sequencing data from all 14 pups confirmed only twoindel sequences reflecting the two alleles arising from the parentalmale and female mouse. This data unequivocally demonstrates that ourone-step genome editing protocol works very efficiently at an earlystage and not beyond the 2 cell stage thus avoiding complex mosaic indelformation. Using our established protocol, we can carry out definedeletions directly in zygotes or carry out define deletion followed byinsertion to expedite the process of generating transgenic mice tohomozygosity in record time.

Example 6 Single Cas9 Cas9 Expression in ES Cells

Reference is made to FIG. 6B.

-   1. A landing pad consisting of a PiggyBac transposon element with    the following features will be targeted into mouse ES cells (e.g.,    129-derived ES cells, such as AB2.1 ES cells; Baylor College of    Medicine, Texas, USA) and selected for on G418. The PiggyBac    transposon element will contain neomycin resistance gene flanked by    loxP and lox2272. It will also have a geneless PGK promoter. In this    example, the landing pad will be targeted into the introgenic region    of Rosa26 gene located on chromosome 6, but it could be targeted    elsewhere. Targeting this landing pad in the Rosa26 gene will    provide a universal ES cell line for precisely inserting any desired    DNA fragment including DNA fragments containing Cas9, mutant Cas9 or    any other gene of interest via RMCE with high efficiency. Targeting    Rosa26 is beneficial since the targeted construct will be inserted    as a single copy (unlike random integration elsewhere) and is    unlikely to produce an unwanted phenotypic effect.

Note. This landing pad can be inserted into any gene in any chromosomeor indeed in any eukaryotic or mammalian cell line, e.g., a human,insect, plant, yeast, mouse, rat, rabbit, rodent, pig, dog, cat, fish,chicken or bird cell line, followed by generation of the respectivetransgenic organism expressing Cas9.

Rosa 26 Locus

Ubiquitous expression of transgene in mouse embryonic stem cell can beachieved by gene targeting to the ROSA26 locus (also known as: gene trapROSA 26 or Gt(ROSA)26) by homologous recombination (Ref. (a) and (b)below). The genomic coordinates for mouse C57BL/6J Rosa26 gene based onEnsemble release 73—September 2013 is: Chromosome 6:113,067,428-113,077,333; reverse strand.

The Rosa26 locus can also be used to as a recipient location to knock-ina transgene. In our example we have use the Rosa26 locus to knock-in thelanding pad vector by targeting through homologous recombination intothe intronic region located between exons 2 and 3 of mouse strain129-derived embryonic stem cells using approx. 3.1 kb homology arms. Thehomology arms were retrieved by recombineering from a BAC Clonegenerated from mouse strain 129. The sequence of the Rosa26 homologyarms used for targeting is given below.

Sequence of Rosa26 5′ homology arm (SEQ ID NO: 23)CACATTTGGTCCTGCTTGAACATTGCCATGGCTCTTAAAGTCTTAATTAAGAATATTAATTGTGTAATTATTGTTTTTCCTCCTTTAGATCATTCCTTGAGGACAGGACAGTGCTTGTTTAAGGCTATATTTCTGCTGTCTGAGCAGCAACAGGTCTTCGAGATCAACATGATGTTCATAATCCCAAGATGTTGCCATTTATGTTCTCAGAAGCAAGCAGAGGCATGATGGTCAGTGACAGTAATGTCACTGTGTTAAATGTTGCTATGCAGTTTGGATTTTTCTAATGTAGTGTAGGTAGAACATATGTGTTCTGTATGAATTAAACTCTTAAGTTACACCTTGTATAATCCATGCAATGTGTATGCAATTACCATTTTAAGTATTGTAGCTTTCTTTGTATGTGAGGATAAAGGTGTTTGTCATAAAATGTTTTGAACATTTCCCCAAAGTTCCAAATTATAAAACCACAACGTTAGAACTTATTTATGAACAATGGTTGTAGTTTCATGCTTTTAAAATGCTTAATTATTCAATTAACACCGTTTGTGTTATAATATATATAAAACTGACATGTAGAAGTGTTTGTCCAGAACATTTCTTAAATGTATACTGTCTTTAGAGAGTTTAATATAGCATGTCTTTTGCAACATACTAACTTTTGTGTTGGTGCGAGCAATATTGTGTAGTCATTTTGAAAAGGAGTCATTTCAATGAGTGTCAGATTGTTTTGAATGTTATTGAACATTTTAAATGCAGACTTGTTCGTGTTTTAGAAAGCAAAACTGTCAGAAGCTTTGAACTAGAAATTAAAAAGCTGAAGTATTTCAGAAGGGAAATAAGCTACTTGCTGTATTAGTTGAAGGAAAGTGTAATAGCTTAGAAAATTTAAAACCATATAGTTGTCATTGCTGAATATCTGGCAGATGAAAAGAAATACTCAGTGGTTCTTTTGAGCAATATAACAGCTTGTTATATTAAAAATTTTCCCCACAGATATAAACTCTAATCTATAACTCATAAATGTTACAAATGGATGAAGCTTACAAATGTGGCTTGACTTGTCACTGTGCTTGTTTTAGTTATGTGAAAGTTTGGCAATAAACCTATGTCCTAAATAGTCAAACTGTGGAATGACTTTTTAATCTATTGGTTTGTCTAGAACAGTTATGTTGCCATTTGCCCTAATGGTGAAAGAAAAAGTGGGGAGTGCCTTGGCACTGTTCATTTGTGGTGTGAACCAAAGAGGGGGGCATGCACTTACACTTCAAACATCCTTTTGAAAGACTGACAAGTTTGGGTCTTCACAGTTGGAATTGGGCATCCCTTTTGTCAGGGAGGGAGGGAGGGAGGGAGGCTGGCTTGTTATGCTGACAAGTGTGATTAAATTCAAACTTTGAGGTAAGTTGGAGGAACTTGTACATTGTTAGGAGTGTGACAATTTGGACTCTTAATGATTTGGTCATACAAAATGAACCTAGACCAACTTCTGGAAGATGTATATAATAACTCCATGTTACATTGATTTCACCTGACTAATACTTATCCCTTATCAATTAAATACAGAAGATGCCAGCCATCTGGGCCTTTTAACCCAGAAATTTAGTTTCAAACTCCTAGGTTAGTGTTCTCACTGAGCTACATCCTGATCTAGTCCTGAAAATAGGACCACCATCACCCCCAAAAAAATCTCAAATAAGATTTATGCTAGTGTTTCAAAATTTTAGGAATAGGTAAGATTAGAAAGTTTTAAATTTTGAGAAATGGCTTCTCTAGAAAGATGTACATAGTGAACACTGAATGGCTCCTAAAGAGCCTAGAAAACTGGTACTGAGCACACAGGACTGAGAGGTCTTTCTTGAAAAGCATGTATTGCTTTACGTGGGTCACAGAAGGCAGGCAGGAAGAACTTGGGCTGAAACTGGTGTCTTAAGTGGCTAACATCTTCACAACTGATGAGCAAGAACTTTATCCTGATGCAAAAACCATCCAAACAAACTAAGTGAAAGGTGGCAATGGATCCCAGGCTGCTCTAGAGGAGGACTTGACTTCTCATCCCATCACCCACACCAGATAGCTCATAGACTGCCAATTAACACCAGCTTCTAGCCTCCACAGGCACCTGCACTGGTACACATAATTTCACACAAACACAGTAAGAAGCCTTCCACCTGGCATGGTATTGCTTATCTTTAGTTCCCAACACTTGGGAGGCAGAGGCCAGCCAGGGCTATGTGACAAAAACCTTGTCTAGAGGAGAAACTTCATAGCTTATTTCCTATTCACGTAACCAGGTTAGCAAAATTTACCAGCCAGAGATGAAGCTAACAGTGTCCACTATATTTGTAGTGTTTTAAGTCAATTTTTTAAATATACTTAATAGAATTAAAGCTATGGTGAACCAAGTACAAACCTGGTGTATTAACTTGAGAACTTAGCATAAAAAGTAGTTCATTTGTTCAGTAAATATTAAATGCTTACTGGCAAAGATTATGTCAGGAACTTGGTAAATGGTGATGAAACAATCATAGTTGTACATCTTGGTTCTGTGATCACCTTGGTTTGAGGTAAAAGTGGTTCCTTTGATCAAGGATGGAATTTTAAGTTTATATTCAATCAATAATGTATTATTTTGTGATTGCAAAATTGCCTATCTAGGGTATAAAACCTTTAAAAATTTCATAATACCAGTTCATTCTCCAGTTACTAATTCCAAAAAGCCACTGACTATGGTGCCAATGTGGATTCTGTTCTCAAAGGAAGGATTGTCTGTGCCCTTTATTCTAATAGAAACATCACACTGAAAATCTAAGCTGAAAGAAGCCAGACTTTCCTAAATAAATAACTTTCCATAAAGCTCAAACAAGGATTACTTTTAGGAGGCACTGTTAAGGAACTGATAAGTAATGAGGTTACTTATATAATGATAGTCCCACAAGACTATCTGAGGAAAAATCAGTACAACTCGAAAACAGAACAACCAGCTAGGCAGGAATAACAGGGCTCCCAAGTCAGGAGGTCTATCCAACACCCTTTTCTGTTGAGGGCCCCAGACCTACATATTGTATACAAACAGGGAGGTGGGTGATTTTAACTCTCCTGAGGTACSequence of Rosa26 3′ homoloty arm (SEQ ID NO: 24)CTTGGTAAATCTTTGTCCTGAGTAAGCAGTACAGTGTACAGTTTACATTTTCATTTAAAGATACATTAGCTCCCTCTACCCCCTAAGACTGACAGGCACTTTGGGGGTGGGGAGGGCTTTGGAAAATAACGCTTCCATACACTAAAAGAGAAATTTCTTTAATTAGGCTTGTTGGTTCCATACATCTACTGGTGTTTCTACTACTTAGTAATATTATAATAGTCACACAAGCATCTTTGCTCTGTTTAGGTTGATATATTTATTTTAAGGCAGATGATAAAACTGTAGATCTTAAGGGATGCTTCTGCTTCTGAGATGATACAAAGAATTTAGACCATAAAACAGTAGGTTGCACAAGCAATAGAATATGGCCTAAAGTGTTCTGACACTTAGAAGCCAAGCAGTGTAGGCTTCTTAAGAAATACCATTACAATCACCTTGCTAGAAATCAAGCATTCTGGAGTGGTCAAGCAGTGTAACCTGTACTGTAAGTTACTTTTCTGCTATTTTTCTCCCAAAGCAAGTTCTTTATGCTGATATTTCCAGTGTTAGGAACTACAAATATTAATAAGTTGTCTTCACTCTTTTCTTTACCAAGGAGGGTCTCTTCCTTCATCTTGATCTGAAGGATGAACAAAGGCTTGAGCAGTGCGCTTTAGAAGATAAACTGCAGCATGAAGGCCCCCGATGTTCACCCAGACTACATGGACCTTTCGCCACACATGTCCCATTCCAGATAAGGCCTGGCACACACAAAAAACATAAGTCATTAGGCTACCAGTCTGATTCTAAAACAACCTAAAATCTTCCCACTTAAATGCTATGGGTGGTGGGTTGGAAAGTTGACTCAGAAAATCACTTGCTGTTTTTAGAGAGGATCTGGGTTCAGTTTCTGATACATTGTGGCTTACAACTATAACTCCAGTTCTAGGGGGTCCATCCAACATCCTCTTCTGTTGAGGGCACCAAATAAATGTATTGTGTACAAACAGGGAGGTGAGTGATTTAACTCTCGTGTATAGTACCTTGGTAAAACATTTCTTGTCCTGAGTAAGCAGTACAGCTCTGCCTGTCCCTGGTCTACAGACACGGCTCATTTCCCGAAGGCAAGCTGGATAGAGATTCCAATTTCTCTTCTTGGATCCCATCCTATAAAAGAAGGTCAAGTTTAATCTATTGCAAAAGGTAAATAGGTAGTTTCTTACATGAGACAAGAACAAATCTTAGGTGTGAAGCAGTCATCTTTTACAGGCCAGAGCCTCTATTCTATGCCAATGAAGGAAACTGTTAGTCCAGTGTTATAGAGTTAGTCCAGTGTATAGTTTTCTATCAGAACACTTTTTTTTTAAACAACTGCAACTTAGCTTATTGAAGACAAACCACGAGTAGAAATCTGTCCAAGAAGCAAGTGCTTCTCAGCCTACAATGTGGAATAGGACCATGTAATGGTACAGTGAGTGAAATGAATTATGGCATGTTTTTCTGACTGAGAAGACAGTACAATAAAAGGTAAACTCATGGTATTTATTTAAAAAGAATCCAATTTCTACCTTTTTCCAAATGGCATATCTGTTACAATAATATCCACAGAAGCAGTTCTCAGTGGGAGGTTGCAGATATCCCACTGAACAGCATCAATGGGCAAACCCCAGGTTGTTTTTCTGTGGAGACAAAGGTAAGATATTTCAATATATTTTCCCAAGCTAATGAGATGGCTCAGCAAATAATGGTACTGGCCATTAAGTCTCATGACCTGAGCTTGATCCTCAGGGACCATGTGGTACAAGGAGAGACCTAAATCCTTCAGTTGGACTTCAATCTTCTACCCTCATGTCCACACACAAATAAATACAATAAAAAACATTCTGCAGTCTGAATTTCTAAAGGTTGTTTTTCTAAAAAGAAATGTTAAAGTAACATAGGAAGAAATATGTCCATAACTGAAATACAAGTTTTTTAAATGGTTAAGACTGGTTTTCAAAGGATGTATGGTTAAGAAAATACCAGGGAAAATGAGCTTACATGTAAAAAAGTGTCTAAAAGGCCAGAGAAATGACCCAGCTGGCAAAGGTGTCTGCCCTAAGCCAGACAAAAGGAATTTGATTCACAGGAAGAAGAGACCCAACTCTCACTAGTTATCCTCTGACTTCCACACCATGACACAGCTCCATGGCACTCTCAGGCCCCCACACATATACAGATATAAACAGAAACCTAATCCACCAGCCTTCAGAAGCAAAGCAATTGGAGGATTTAAACAGGCCATGGCTACTAATAGAGATAACTGGTAGTTTAAAAGTTATGGTAATGACTTTCATGCTTCTTTCAACTCATATTGTTCTAAATAATTAATTTGGTTTTTCAAGGCAGGGTTTCTCTGTGTAGTTCTGGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTTGAACTCAGATCCATCTGCCTCTGGAATAAGGGCACGTGCGTGCCTTTTCTACATAACAAAACCTATACTATAACAAAACCTATACCATACTGTACCGTTTTGGGAAAAGACAAAAAATAATGAACAAAAAAGGAGAAATAACATTCCAATAAAGTATGGAAATGGTAGTTAAATTAATTACAAATGTTTTTCAGTAAATTAGATGTGACTTCTCATACTGTTCATTTGGCTATAATGATACCACAAAGCACTGGGGGTGAATAATAATTCCAAGTCAGTAGGGAGAGAGACTTGAAAAGATGCAATGCAATCATTGAAGTTAAACTTACCCATCTTTAATCTGGCTCTTAGTCAATAGAGATGAGATGTTATTTGCTGCTCTGTTCACTGCCAGTGGGTTATTGTCCCCAGCAATATGGTAACAGTGAGACCACTCAGTAGCCCCCTATGAGACAGGAGTGTTGGTTAAACATGCCACAAGAGAAAAGGGAAAAGTCACTATGGCCAACTCTCAGTAACATGGCAATCCGTGCCATTCATTTCCTTGCCAGAAATGTCTTCCCTGTTCTTCTGCCTACTGAACTTTCACCCACTAGAAATGTGGCTCCAATGTCATCCACTATGACATCAATGTCAGCGCTAGAAGCACTTTGCACACCTCTGTTGCTGACTTAG

REFERENCE

-   a) Pablo Perez-Pinera, David G. Ousterout, Matthew T. Brown and    Charles A. Gersbach (2012) Gene targeting to the ROSA26 locus    directed by engineered zinc finger nucleases. Nucleic Acids    Research, 2012, Vol. 40, No. 8 3741-3752-   b) Peter Hohenstein, Joan Slight, Derya Deniz Ozdemir, Sally F Burn,    Rachel Berry and Nicholas D Hastie (2008) High-efficiency Rosa26    knock-in vector construction for Cre-regulated overexpression and    RNAi. PathoGenetics 2008, 1:3-   2. A recombinase mediated cassette exchange (RMCE)-enabled vector    containing a promoterless puromycin-delta-tk with in-frame fusion of    T2A at the C-terminus following by either Cas9 or mutant Cas9    nucleotide sequence and a series of unique restriction sites flanked    by loxP and lox2272 will allow for the direct targeting of this    vector into the landing pad by Cre-mediated RMCE. As is known, T2A    allows ribosomal skipping during translation. The insertion of the    coding sequence of T2A between two genes results in two products    (one gene, one transcript but two proteins expressed, in this case    the Cas9 and selection marker). ES clones containing the correctly    inserted DNA fragment can be directly selected on puromycin. This    approach also advantageously ensures single copy expression of Cas9    as suppose to a random integration or transient expression approach.    Insertion of the RMCE enabled vector into the desired locus    containing the landing pad can be selected directly as the PGK    promoter in the landing pad will drive the transcription of the    promoterless Puro-Delta-Tk and Cas9. Since the Puro-delta-Tk is in    the same transcriptional unit as Cas9, ES clones selected on    puromycin will ensure expression of Cas9.-   3. The above strategy allows for three separate approaches to    express the sgRNA designed for disrupting (mutation through indel    formation, deletion or deletion followed by insertion) gene of    interest.    -   a. The above ES cell line containing Cas9 can be used for        generating transgenic mice with either constitutively expressed        Cas9 or modified for inducible Cas9 expression or indeed tissue        specific Cas9 expression for example expression of Cas9 at an        embryo stage using Nanog-, PouSfl- or SoxB promoter-specific        Cas9 expression. Such derived mouse line expressing Cas9 can be        used for genome editing in a streamline fashion whereby in vitro        transcribed sgRNA can be easily injected into embryos obtained        from such transgenic mice. This will enhance the efficiency of        generating mouse lines with the desired homozygous genotype and        thus will dramatically reduce the number of animals required.    -   b. sgRNA can be transfected directly into the ES cells        expressing Cas9 and thus avoids the requirement for cloning into        the RMCE enabled vector single or multiple sgRNA. This approach        will allow multiple sgRNA to be inserted into the ES cells        simultaneously very rapidly.    -   c. Multiple sgRNA can be cloned directly into the multiple        cloning site of the RMCE enabled vector (ie, using a plurality        of different restriction endonuclease sites) to allow single        copy expression of the guide-RNA. This approach may be useful        for limiting off-target effects particularly relevant for those        genes with high sequence homology within the genome.-   4. ES cells expressing Cas9 and sgRNA can be selected for directly    on medium containing puromycin. Selection on puromycin for 4-6 days    will allow for the desired location to be mutated or disrupted and    the advantage of manipulating ES cells is that individual clones can    be analysed by PCR followed by sequencing for the desired mutation.    Only correctly mutated ES cell clones can be processed further    whereby inserted DNA element introduced through insertion of the    landing pad and the subsequent insertion of the RMCE vector can be    completely removed leaving the ES cell devoid of any alteration    other than the intended mutation induced by the action of Cas9 and    the sgRNA. This can be done through transiently expressing PBase    transposon followed by selection on FIAU. Removal of the    constitutively expressed Cas9 with only the minimal length of time    required to induce mutation in the presence of sgRNA will reduce or    eliminate the possibility of Cas9 inducing unwanted mutations.-   5. ES Clones containing the desired mutation can be injected into    blastocyst to generate transgenic mice.

TABLE 1 PAM conservation in reeats and leaders for various CRISPR types(reproduced from Short motif sequences determine the targets of theprokaryotic CRISPR defence system F. J. M. Molica, C. Diez-Villaserior,J. Garcia-Martinez, C. Almendros Microbiology(2009), 155, 733-740)Genomes* PAM CRISPR Consensus^(†) Leaders^(‡) Group 1 Mth NGGATTTCAATCCCATTTTGGTCTGATTTTAAC AGGGCGGATT ATGGCCAATT Lmo WGGATTTACATTTCAHAATAAGTARYTAAAAC CCACTAACTT CCGCTCTATT Group 2 Eco CWTCGGTTTATCCCCGCTGGCGCGGGGAACWC TCTAAACATA TCTAAAAGTA Pae CTTCGGTTCATCCCCACRCMYGTGGGGAACAC ACTTACCGTA CCTTACCGTA Group 3 Spy GAAATTTCAATCCACTCACCCATGAAGGGTGAGAC TGCGCCAAAT Xan GAAGTTTCAATCCACGCGCCCGTGAGGRCGCGAC CCCCCCTTAG GCCGCCAGCA Group 4 She GGTTTCTAAGCCGCCTGTGCGGCGGTGAAC AATAGCTTAT TGTAGAATAA Pae GGTTTCTTAGCTGCCTATACGGCAGTGAAC TAGCTCCGAA TAGACCAAAA Ype GGTTTCTAAGCTGCCTGTGCGGCAGTGAAC GTAAGATAAT Group 7 Sso NGGCTTTCAATTCTATAAGAGATTATC TGAGGGTTTA Mse NGG CTTTCAACTCTATAGGAGATTAACTGATACCTTT TGAAACTTTT TGACACTCTT Group 10 Str NGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC CTCGTAGACT CTCGTAGAAA Lis NGGGTTTTAGAGCTATGTTATTTTGAATGCTAMCAAAAC CTCGCAGAAT CTCGTAGAAT *Genomes areabbreviated according to the denominations of the species or generacarrying the corresponding CRISPR arrays: Mth, M. thermautotrophicus;Lmo, L. monocytogenes; Eco, E. coli; Pae, P. aeruginosa; Spy, S.pyogenes; Xan, Xanthomonas spp.; She, Shewanella spp,; Ype, Y. pestis,Sso, S. solfataricus; Mse, M. sedula; Str, Streptococcus spp.; Lis,Listeria spp. ^(†)Sequences matching the PAM are underlined.^(‡)Representative CRISPR array proximal Leader sequences. Nucleotidesmatching the PAM are underlined.

SEQ ID NOs for the sequences in Table 1 are set out in the table below.

SEQ SEQ dfd Genomes* PAM CRISPR Consens^(†) ID NO. Leaders^(‡) ID NO.Group 1 Mth NGG ATTTCAATCCCATTTTGGTCTGATTTTAAC 25 AGGGCGGATT 38ATGGCCAATT 39 Lmo WGG ATTTACATTTCAHAATAAGTARYTAAAAC 26 CCACTAACTT 40CCGCTCTATT 41 Group 2 Eco CWT CGGTTTATCCCCGCTGGCGCGGGGAACWC 27TCTAAACATA 42 TCTAAAAGTA 43 Pae CTT CGGTTCATCCCCACRCMYGTGGGGAACAC 28ACTTACCGTA 44 CCTTACCGTA 45 Group 3 Spy GAAATTTCAATCCACTCACCCATGAAGGGTGAGAC 29 TGCGCCAAAT 46 Xan GAAGTTTCAATCCACGCGCCCGTGAGGRCGCGAC 30 CCCCCCTTAG 47 GCCGCCAGCA 48 Group 4She GG TTTCTAAGCCGCCTGTGCGGCGGTGAAC 31 AATAGCTTAT 49 TGTAGAATAA 50 PaeGG TTTCTTAGCTGCCTATACGGCAGTGAAC 32 TAGCTCCGAA 51 TAGACCAAAA 52 Ype GGTTTCTAAGCTGCCTGTGCGGCAGTGAAC 33 GTAAGATAAT 53 Group 7 Sso NGGCTTTCAATTCTATAAGAGATTATC 34 TGAGGGTTTA 54 Mse NGGCTTTCAACTCTATAGGAGATTAAC 35 TGATACCTTT 55 TGAAACTTTT 56 TGACACTCTT 57Group 10 Str NGG GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC 36 CTCGTAGACT 58CTCGTAGAAA 59 Lis NGG GTTTTAGAGCTATGTTATTTTGAATGCTAMCAAAAC 37 CTCGCAGAAT60 CTCGTAGAAT 61

TABLE 2 CRISPR-Associated Endonucleases [Gene ID numbers refer to genesin the NCBI Gene Database as at September 2013; all sequence informationrelating to the gene IDs below is incorporated herein by reference forpossible use in the present invention] 1. Plav_0099 CRISPR-associatedendonuclease Csn1 family protein [Parvibaculum lavamentivorans DS-1]Other Aliases: Plav_0099 Genomic context: Chromosome Annotation:NC_009719.1 (105795 . . . 108908, complement) ID: 5454634 SEQ ID NO: 622. FTN_0757 membrane protein [Francisella novicida U112] Other Aliases:FTN_0757 Genomic context: Chromosome Annotation: NC_008601.1 (810052 . .. 814941) ID: 4548251 SEQ ID NO: 63 3. Cj1523c CRISPR-associated protein[Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819] OtherAliases: Cj1523c Genomic context: Chromosome Annotation: NC_002163.1(1456880 . . . 1459834, complement) ID: 905809 SEQ ID NO: 64 4. mcrArestriction endonuclease [Bifidobacterium longum DJO10A] Other Aliases:BLD_1902 Genomic context: Chromosome Annotation: NC_010816.1 (2257993 .. . 2261556) ID: 6362834 SEQ ID NO: 65 5. MGA_0519 Csn1 familyCRISPR-associated protein [Mycoplasma gallisepticum str. R(low)] OtherAliases: MGA_0519 Genomic context: Chromosome Annotation: NC_004829.2(919248 . . . 923060) ID: 1089911 SEQ ID NO: 66 6. Emin_0243CRISPR-associated endonuclease Csn1 family protein [Elusimicrobiumminutum Pei191] Other Aliases: Emin_0243 Genomic context: ChromosomeAnnotation: NC_010644.1 (261119 . . . 264706) ID: 6263045 SEQ ID NO: 677. FTW_1427 CRISPR-associated large protein [Francisella tularensissubsp. tularensis WY96-3418] Other Aliases: FTW_1427 Genomic context:Chromosome Annotation: NC_009257.1 (1332426 . . . 1335803, complement)ID: 4958852 SEQ ID NO: 68 8. SMA_1444 CRISPR-associated protein, Csn1family [Streptococcus macedonicus ACA-DC 198] Other Aliases: SMA_1444Annotation: NC_016749.1 (1418337 . . . 1421729, complement) ID: 11601419SEQ ID NO: 69 9. SSUST3_1318 CRISPR-associated protein, Csn1 family[Streptococcus suis ST3] Other Aliases: SSUST3_1318 Genomic context:Chromosome Annotation: NC_015433.1 (1323872 . . . 1327240, complement)ID: 10491484 SEQ ID NO: 70 10. cas5 CRISPR-associated protein, Csn1family [Streptococcus gallolyticus UCN34] Other Aliases: GALLO_1439Genomic context: Chromosome Annotation: NC_013798.1 (1511433 . . .1514825, complement) ID: 8776949 SEQ ID NO: 71 11. GALLO_1446CRISPR-associated protein [Streptococcus gallolyticus UCN34] OtherAliases: GALLO_1446 Genomic context: Chromosome Annotation: NC_013798.1(1518984 . . . 1523110, complement) ID: 8776185 SEQ ID NO: 72 12. csn1CRISPR-associated endonuclease Csn1 [Bifidobacterium dentium Bd1] OtherAliases: BDP_1254 Genomic context: Chromosome Annotation: NC_013714.1(1400576 . . . 1403992, complement) ID: 8692053 SEQ ID NO: 73 13.NMO_0348 putative CRISPR-associated protein [Neisseria meningitidesalpha14] Other Aliases: NMO_0348 Genomic context: Chromosome Annotation:NC_013016.1 (369547 . . . 372795, complement) ID: 8221228 SEQ ID NO: 7414. csn1 CRISPR-Associated Protein Csn1 [Streptococcus equi subsp.zooepidemicus MGCS10565] Other Aliases: Sez_1330 Genomic context:Chromosome Annotation: NC_011134.1 (1369339 . . . 1373385, complement)ID: 6762114 SEQ ID NO: 75 15. csn1 CRISPR-associated endonuclease Csn1family protein [Streptococcus gordonii str. Challis substr. CH1] OtherAliases: SGO_1381 Genomic context: Chromosome Annotation: NC_009785.1(1426750 . . . 1430160, complement) ID: 5599802 SEQ ID NO: 76 16.M28_Spy0748 cytoplasmic protein [Streptococcus pyogenes MGAS6180] OtherAliases: M28_Spy0748 Genomic context: Chromosome Annotation: NC_007296.1(771231 . . . 775337) ID: 3573516 SEQ ID NO: 77 17. SGGBAA2069_c14690CRISPR-associated protein [Streptococcus gallolyticus subsp.gallolyticus ATCC BAA-2069] Other Aliases: SGGBAA2069_c14690 Genomiccontext: Chromosome Annotation: NC_015215.1 (1520905 . . . 1525017,complement) ID: 10295470 SEQ ID NO: 78 18. SAR116_2544 CRISPR-associatedprotein, Csn1 family [Candidatus Puniceispirillum marinum IMCC1322]Other Aliases: SAR116_2544 Genomic context: Chromosome Annotation:NC_014010.1 (2748992 . . . 2752099) ID: 8962895 SEQ ID NO: 79 19.TDE0327 CRISPR-associated Cas5e [Treponema denticola ATCC 35405] OtherAliases: TDE0327 Genomic context: Chromosome Annotation: NC_002967.9(361021 . . . 365208) ID: 2741543 SEQ ID NO: 80 20. csn1CRISPR-associated protein [Streptococcus pasteurianus ATCC 43144] OtherAliases: SGPB_1342 Genomic context: Chromosome Annotation: NC_015600.1(1400035 . . . 1403427, complement) ID: 10753339 SEQ ID NO: 81 21. cas9CRISPR-associated protein [Corynebacterium ulcerans BR-AD22] OtherAliases: CULC22_00031 Genomic context: Chromosome Annotation:NC_015683.1 (30419 . . . 33112, complement) ID: 10842578 SEQ ID NO: 8222. MGAS2096_Spy0843 putative cytoplasmic protein [Streptococcuspyogenes MGAS2096] Other Aliases: MGAS2096_Spy0843 Genomic context:Chromosome Annotation: NC_008023.1 (813084 . . . 817190) ID: 4066021 SEQID NO: 83 23. MGAS9429_Spy0885 cytoplasmic protein [Streptococcuspyogenes MGAS9429] Other Aliases: MGAS9429_Spy0885 Genomic context:Chromosome Annotation: NC_008021.1 (852508 . . . 856614) ID: 4061575 SEQID NO: 84 24. AZL_009000 CRISPR-associated protein, Csn1 family[Azospirillum sp. B510] Other Aliases: AZL_009000 Genomic context:Chromosome Annotation: NC_013854.1 (1019522 . . . 1023028, complement)ID: 8789261 SEQ ID NO: 85 25. EUBREC_1713 contains RuvC-like nucleaseand HNH-nuclease domains [Eubacterium rectale ATCC 33656] Other Aliases:EUBREC_1713 Other Designations: CRISPR-system related protein Genomiccontext: Chromosome Annotation: NC_012781.1 (1591112 . . . 1594456) ID:7963668 SEQ ID NO: 86 26. Alide2_0194 CRISPR-associated protein, Csn1family [Alicycliphilus denitrificans K601] Other Aliases: Alide2_0194Genomic context: Chromosome Annotation: NC_015422.1 (218107 . . .221196) ID: 10481210 SEQ ID NO: 87 27. Alide_0205 crispr-associatedprotein, csn1 family [Alicycliphilus denitrificans BC] Other Aliases:Alide_0205 Genomic context: Chromosome Annotation: NC_014910.1 (228371 .. . 231460) ID: 10102228 SEQ ID NO: 88 28. STER_1477 CRISPR-system-likeprotein [Streptococcus thermophilus LMD-9] Other Aliases: STER_1477Genomic context: Chromosome Annotation: NC_008532.1 (1379975 . . .1384141, complement) ID: 4437923 SEQ ID NO: 89 29. STER_0709CRISPR-system-like protein [Streptococcus thermophilus LMD-9] OtherAliases: STER_0709 Genomic context: Chromosome Annotation: NC_008532.1(643235 . . . 646600) ID: 4437391 SEQ ID NO: 90 30. cas9CRISPR-associated protein [Corynebacterium diphtheriae 241] OtherAliases: CD241_2102 Genomic context: Chromosome Annotation: NC_016782.1(2245769 . . . 2248399) ID: 11674395 SEQ ID NO: 91 31. cas3CRISPR-associated endonuclease [Corynebacterium diphtheriae 241] OtherAliases: CD241_0034 Genomic context: Chromosome Annotation: NC_016782.1(35063 . . . 38317) ID: 11672999 SEQ ID NO: 92 32. Corgl_1738CRISPR-associated protein, Csn1 family [Coriobacterium glomerans PW2]Other Aliases: Corgl_1738 Genomic context: Chromosome Annotation:NC_015389.1 (2036091 . . . 2040245) ID: 10439994 SEQ ID NO: 93 33.Fluta_3147 CRISPR-associated protein, Csn1 family [Fluviicola taffensisDSM 16823] Other Aliases: Fluta_3147 Genomic context: ChromosomeAnnotation: NC_015321.1 (3610221 . . . 3614597, complement) ID: 10398516SEQ ID NO: 94 34. Acav_0267 CRISPR-associated protein, Csn1 family[Acidovorax avenae subsp. avenae ATCC 19860] Other Aliases: Acav_0267Genomic context: Chromosome Annotation: NC_015138.1 (295839 . . .298976) ID: 10305168 SEQ ID NO: 95 35. NAL212_2952 CRISPR-associatedprotein, Csn1 family [Nitrosomonas sp. AL212] Other Aliases: NAL212_2952Genomic context: Chromosome Annotation: NC_015222.1 (2941806 . . .2944940, complement) ID: 10299493 SEQ ID NO: 96 36. SpiBuddy_2181CRISPR-associated protein, Csn1 family [Sphaerochaeta globosa str.Buddy] Other Aliases: SpiBuddy_2181 Genomic context: ChromosomeAnnotation: NC_015152.1 (2367952 . . . 2371491, complement) ID: 10292274SEQ ID NO: 97 37. Tmz1t_2411 HNH endonuclease [Thauera sp. MZ1T] OtherAliases: Tmz1t_2411 Genomic context: Plasmid pTha01 Annotation:NC_011667.1 (75253 . . . 76200, complement) ID: 7094333 SEQ ID NO: 9838. Gdia_0342 Csn1 family CRISPR-associated protein [Gluconacetobacterdiazotrophicus PAI 5] Other Aliases: Gdia_0342 Genomic context:Chromosome Annotation: NC_011365.1 (382737 . . . 385748) ID: 6973736 SEQID NO: 99 39. JJD26997_1875 CRISPR-associated Cas5e family protein[Campylobacter jejuni subsp. doylei 269.97] Other Aliases: JJD26997_1875Genomic context: Chromosome Annotation: NC_009707.1 (1656109 . . .1659063, complement) ID: 5389688 SEQ ID NO: 100 40. Asuc_0376CRISPR-associated endonuclease Csn1 family protein [Actinobacillussuccinogenes 130Z] Other Aliases: Asuc_0376 Genomic context: ChromosomeAnnotation: NC_009655.1 (431928 . . . 435116) ID: 5348478 SEQ ID NO: 10141. Veis_1230 CRISPR-associated endonuclease Csn1 family protein[Verminephrobacter eiseniae EF01-2] Other Aliases: Veis_1230 Genomiccontext: Chromosome Annotation: NC_008786.1 (1365979 . . . 1369185) ID:4695198 SEQ ID NO: 102 42. MGAS10270_Spy0886 putative cytoplasmicprotein [Streptococcus pyogenes MGAS10270] Other Aliases:MGAS10270_Spy0886 Genomic context: Chromosome Annotation: NC_008022.1(844446 . . . 848552) ID: 4063984 SEQ ID NO: 103 43. gbs0911hypothetical protein [Streptococcus agalactiae NEM316] Other Aliases:gbs0911 Genomic context: Chromosome Annotation: NC_004368.1 (945801 . .. 949946) ID: 1029893 SEQ ID NO: 104 44. NMA0631 hypothetical protein[Neisseria meningitidis Z2491] Other Aliases: NMA0631 Genomic context:Chromosome Annotation: NC_003116.1 (610868 . . . 614116, complement) ID:906626 SEQ ID NO: 105 45. Ccan_14650 hypothetical protein[Capnocytophaga canimorsus Cc5] Other Aliases: Ccan_14650 Genomiccontext: Chromosome Annotation: NC_015846.1 (1579873 . . . 1584165,complement) ID: 10980451 SEQ ID NO: 106 46. lpp0160 hypothetical protein[Legionella pneumophila str. Paris] Other Aliases: lpp0160 Genomiccontext: Chromosome Annotation: NC_006368.1 (183831 . . . 187949) ID:3118625 SEQ ID NO: 107 47. Cbei_2080 hypothetical protein [Clostridiumbeijerinckii NCIMB 8052] Other Aliases: Cbei_2080 Genomic context:Chromosome Annotation: NC_009617.1 (2422056 . . . 2423096) ID: 5296367SEQ ID NO: 108 48. MMOB0330 hypothetical protein [Mycoplasma mobile163K] Other Aliases: MMOB0330 Genomic context: Chromosome Annotation:NC_006908.1 (45652 . . . 49362, complement) ID: 2807677 SEQ ID NO: 10949. MGF_5203 Csn1 family CRISPR-associated protein [Mycoplasmagallisepticum str. F] Other Aliases: MGF_5203 Genomic context:Chromosome Annotation: NC_017503.1 (888602 . . . 892411) ID: 12397088SEQ ID NO: 110 50. MGAH_0519 Csn1 family CRISPR-associated protein[Mycoplasma gallisepticum str. R(high)] Other Aliases: MGAH_0519 Genomiccontext: Chromosome Annotation: NC_017502.1 (918476 . . . 922288) ID:12395725 SEQ ID NO: 111 51. Smon_1063 CRISPR-associated protein, Csn1family [Streptobacillus moniliformis DSM 12112] Other Aliases: Smon_1063Genomic context: Chromosome Annotation: NC_013515.1 (1159048 . . .1162827, complement) ID: 8600791 SEQ ID NO: 112 52. Spy49_0823hypothetical protein [Streptococcus pyogenes NZ131] Other Aliases:Spy49_0823 Genomic context: Chromosome Annotation: NC_011375.1 (821210 .. . 825316) ID: 6985827 SEQ ID NO: 113 53. C8J_1425 hypothetical protein[Campylobacter jejuni subsp. jejuni 81116] Other Aliases: C8J_1425Genomic context: Chromosome Annotation: NC_009839.1 (1442672 . . .1445626, complement) ID: 5618449 SEQ ID NO: 114 54. FTF0584 hypotheticalprotein [Francisella tularensis subsp. tularensis FSC198] Other Aliases:FTF0584 Genomic context: Chromosome Annotation: NC_008245.1 (601115 . .. 604486) ID: 4200457 SEQ ID NO: 115 55. FTT_0584 hypothetical protein[Francisella tularensis subsp. tularensis SCHU S4] Other Aliases:FTT_0584 Genomic context: Chromosome Annotation: NC_006570.2 (601162 . .. 604533) ID: 3191177 SEQ ID NO: 116 56. csn1 CRISPR-associated protein[Streptococcus dysgalactiae subsp. equisimilis RE378] Other Aliases:GGS_1116 Annotation: NC_018712.1 (1169559 . . . 1173674, complement) ID:13799322 SEQ ID NO: 117 57. SMUGS5_06270 CRISPR-associated protein csn1[Streptococcus mutans GS-5] Other Aliases: SMUGS5_06270 Genomic context:Chromosome Annotation: NC_018089.1 (1320641 . . . 1324678, complement)ID: 13299050 SEQ ID NO: 118 58. Y1U_C1412 Csn1 [Streptococcusthermophilus MN-ZLW-002] Other Aliases: Y1U_C1412 Genomic context:Chromosome Annotation: NC_017927.1 (1376653 . . . 1380819, complement)ID: 12977193 SEQ ID NO: 119 59. Y1U_C0633 CRISPR-system-like protein[Streptococcus thermophilus MN-ZLW-002] Other Aliases: Y1U_C0633 Genomiccontext: Chromosome Annotation: NC_017927.1 (624274 . . . 627639) ID:12975630 SEQ ID NO: 120 60. SALIVA_0715 CRISPR-associated endonuclease,Csn1 family [Streptococcus salivarius JIM8777] Other Aliases:SALIVA_0715 Annotation: NC_017595.1 (708034 . . . 711417) ID: 12910728SEQ ID NO: 121 61. csn1 CRISPR-associated protein csn1 [Streptococcusmutans LJ23] Other Aliases: SMULJ23_0701 Annotation: NC_017768.1 (751695. . . 755732) ID: 12898085 SEQ ID NO: 122 62. RIA_1455 CRISPR-associatedprotein, SAG0894 [Riemerella anatipestifer RA-GD] Other Aliases:RIA_1455 Genomic context: Chromosome Annotation: NC_017569.1 (1443996 .. . 1448198) ID: 12613647 SEQ ID NO: 123 63. STND_0658 CRISPR-associatedendonuclease, Csn1 family [Streptococcus thermophilus ND03] OtherAliases: STND_0658 Genomic context: Chromosome Annotation: NC_017563.1(633621 . . . 636986) ID: 12590813 SEQ ID NO: 124 64. RA0C_1034 putativeBCR [Riemerella anatipestifer ATCC 11845 = DSM 15868] Other Aliases:RA0C_1034 Genomic context: Chromosome Annotation: NC_017045.1 (1023494 .. . 1026931, complement) ID: 11996006 SEQ ID NO: 125 65. Sinf_1255CRISPR-associated protein, SAG0894 family [Streptococcus infantariussubsp. infantarius CJ18] Other Aliases: Sinf_1255 Genomic context:Chromosome Annotation: NC_016826.1 (1276484 . . . 1280611, complement)ID: 11877786 SEQ ID NO: 126 66. Nitsa_1472 CRISPR-associated protein,csn1 family [Nitratifractor salsuginis DSM 16511] Other Aliases:Nitsa_1472 Genomic context: Chromosome Annotation: NC_014935.1 (1477331. . . 1480729) ID: 10148263 SEQ ID NO: 127 67. NLA_17660 hypotheticalprotein [Neisseria lactamica 020-06] Other Aliases: NLA_17660 Genomiccontext: Chromosome Annotation: NC_014752.1 (1890078 . . . 1893326) ID:10006697 SEQ ID NO: 128 68. SmuNN2025_0694 hypothetical protein[Streptococcus mutans NN2025] Other Aliases: SmuNN2025_0694 Genomiccontext: Chromosome Annotation: NC_013928.1 (737258 . . . 741295) ID:8834629 SEQ ID NO: 129 69. SDEG_1231 hypothetical protein [Streptococcusdysgalactiae subsp. equisimilis GGS_124] Other Aliases: SDEG_1231Chromosome: 1 Annotation: Chromosome 1NC_012891.1 (1176755 . . .1180870, complement) ID: 8111553 SEQ ID NO: 130 70. NMCC_0397hypothetical protein [Neisseria meningitidis 053442] Other Aliases:NMCC_0397 Genomic context: Chromosome Annotation: NC_010120.1 (402733 .. . 405981, complement) ID: 5796426 SEQ ID NO: 131 71. SAK_1017hypothetical protein [Streptococcus agalactiae A909] Other Aliases:SAK_1017 Genomic context: Chromosome Annotation: NC_007432.1 (980303 . .. 984415) ID: 3686185 SEQ ID NO: 132 72. M5005_Spy_0769 hypotheticalprotein [Streptococcus pyogenes MGAS5005] Other Aliases: M5005_Spy_0769Genomic context: Chromosome Annotation: NC_007297.1 (773340 . . .777446) ID: 3572134 SEQ ID NO: 133 73. MS53_0582 hypothetical protein[Mycoplasma synoviae 53] Other Aliases: MS53_0582 Genomic context:Chromosome Annotation: NC_007294.1 (684155 . . . 688099) ID: 3564051 SEQID NO: 134 74. DIP0036 hypothetical protein [Corynebacterium diphtheriaeNCTC 13129] Other Aliases: DIP0036 Genomic context: ChromosomeAnnotation: NC_002935.2 (34478 . . . 37732) ID: 2650188 SEQ ID NO: 13575. WS1613 hypothetical protein [Wolinella succinogenes DSM 1740] OtherAliases: WS1613 Genomic context: Chromosome Annotation: NC_005090.1(1525628 . . . 1529857) ID: 2553552 SEQ ID NO: 136 76. PM1127hypothetical protein [Pasteurella multocida subsp. multocida str. Pm70]Other Aliases: PM1127 Genomic context: Chromosome Annotation:NC_002663.1 (1324015 . . . 1327185, complement) ID: 1244474 SEQ ID NO:137 77. SPs1176 hypothetical protein [Streptococcus pyogenes SSI-1]Other Aliases: SPs1176 Genomic context: Chromosome Annotation:NC_004606.1 (1149610 . . . 1153716, complement) ID: 1065374 SEQ ID NO:138 78. SMU_1405c hypothetical protein [Streptococcus mutans UA159]Other Aliases: SMU_1405c, SMU.1405c Genomic context: ChromosomeAnnotation: NC_004350.2 (1330942 . . . 1334979, complement) ID: 1028661SEQ ID NO: 139 79. lin2744 hypothetical protein [Listeria innocuaClip11262] Other Aliases: lin2744 Genomic context: ChromosomeAnnotation: NC_003212.1 (2770707 . . . 2774711, complement) ID: 1131597SEQ ID NO: 140 80. csn1B CRISPR-associated protein [Streptococcusgallolyticus subsp. gallolyticus ATCC 43143] Other Aliases: SGGB_1441Annotation: NC_017576.1 (1489111 . . . 1493226, complement) ID: 12630646SEQ ID NO: 141 81. csn1A CRISPR-associated protein [Streptococcusgallolyticus subsp. gallolyticus ATCC 43143] Other Aliases: SGGB_1431Annotation: NC_017576.1 (1480439 . . . 1483831, complement) ID: 12630636SEQ ID NO: 142 82. cas9 CRISPR-associated protein [Corynebacteriumulcerans 809] Other Aliases: CULC809_00033 Genomic context: ChromosomeAnnotation: NC_017317.1 (30370 . . . 33063, complement) ID: 12286148 SEQID NO: 143 83. GDI_2123 hypothetical protein [Gluconacetobacterdiazotrophicus PAI 5] Other Aliases: GDI_2123 Genomic context:Chromosome Annotation: NC_010125.1 (2177083 . . . 2180235) ID: 5792482SEQ ID NO: 144 84. Nham_4054 hypothetical protein [Nitrobacterhamburgensis X14] Other Aliases: Nham_4054 Genomic context: Plasmid 1Annotation: NC_007959.1 (13284 . . . 16784, complement) ID: 4025380 SEQID NO: 145 85. str0657 hypothetical protein [Streptococcus thermophilusCNRZ1066] Other Aliases: str0657 Genomic context: Chromosome Annotation:NC_006449.1 (619189 . . . 622575) ID: 3165636 SEQ ID NO: 146 86. stu0657hypothetical protein [Streptococcus thermophilus LMG 18311] OtherAliases: stu0657 Genomic context: Chromosome Annotation: NC_006448.1(624007 . . . 627375) ID: 3165000 SEQ ID NO: 147 87. SpyM3_0677hypothetical protein [Streptococcus pyogenes MGAS315] Other Aliases:SpyM3_0677 Genomic context: Chromosome Annotation: NC_004070.1 (743040 .. . 747146) ID: 1008991 SEQ ID NO: 148 88. HFMG06CAA_5227 Csn1 familyCRISPR-associated protein [Mycoplasma gallisepticum CA06_2006.052-5-2P]Other Aliases: HFMG06CAA_5227 Genomic context: Chromosome Annotation:NC_018412.1 (895338 . . . 899147) ID: 13464859 SEQ ID NO: 149 89.HFMG01WIA_5025 Csn1 family CRISPR-associated protein [Mycoplasmagallisepticum WI01_2001.043-13-2P] Other Aliases: HFMG01WIA_5025 Genomiccontext: Chromosome Annotation: NC_018410.1 (857648 . . . 861457) ID:13463863 SEQ ID NO: 150 90. HFMG01NYA_5169 Csn1 family CRISPR-associatedprotein [Mycoplasma gallisepticum NY01_2001.047-5-1P] Other Aliases:HFMG01NYA_5169 Genomic context: Chromosome Annotation: NC_018409.1(883511 . . . 887185) ID: 13462600 SEQ ID NO: 151 91. HFMG96NCA_5295Csn1 family CRISPR-associated protein [Mycoplasma gallisepticumNC96_1596-4-2P] Other Aliases: HFMG96NCA_5295 Genomic context:Chromosome Annotation: NC_018408.1 (904664 . . . 908473) ID: 13462279SEQ ID NO: 152 92. HFMG95NCA_5107 Csn1 family CRISPR-associated protein[Mycoplasma gallisepticum NC95_13295-2-2P] Other Aliases: HFMG95NCA_5107Genomic context: Chromosome Annotation: NC_018407.1 (871783 . . .875592) ID: 13461469 SEQ ID NO: 153 93. MGAS10750_Spy0921 hypotheticalprotein [Streptococcus pyogenes MGAS10750] Other Aliases:MGAS10750_Spy0921 Genomic context: Chromosome Annotation: NC_008024.1(875719 . . . 879834) ID: 4066656 SEQ ID NO: 154 94. XAC3262hypothetical protein [Xanthomonas axonopodis pv. citri str. 306] OtherAliases: XAC3262 Genomic context: Chromosome Annotation: NC_003919.1(3842310 . . . 3842765) ID: 1157333 SEQ ID NO: 155 95. SSUST1_1305CRISPR-system-like protein [Streptococcus suis ST1] Other Aliases:SSUST1_1305 Genomic context: Chromosome Annotation: NC_017950.1 (1293105. . . 1297250, complement) ID: 13017849 SEQ ID NO: 156 96. SSUD9_1467CRISPR-associated protein, Csn1 family [Streptococcus suis D9] OtherAliases: SSUD9_1467 Genomic context: Chromosome Annotation: NC_017620.1(1456318 . . . 1459686, complement) ID: 12718289 SEQ ID NO: 157 97.BBta_3952 hypothetical protein [Bradyrhizobium sp. BTAi1] Other Aliases:BBta_3952 Genomic context: Chromosome Annotation: NC_009485.1 (4149455 .. . 4152649, complement) ID: 5151538 SEQ ID NO: 158 98. CIY_03670CRISPR-associated protein, Csn1 family [Butyrivibrio fibrisolvens 16/4]Other Aliases: CIY_03670 Annotation: NC_021031.1 (309663 . . . 311960,complement) ID: 15213189 SEQ ID NO: 159 99. A11Q_912 CRISPR-associatedprotein, Csn1 family [Bdellovibrio exovorus JSS] Other Aliases: A11Q_912Genomic context: Chromosome Annotation: NC_020813.1 (904781 . . .907864, complement) ID: 14861475 SEQ ID NO: 160 100. MCYN0850 Csn1family CRISPR-associated protein [Mycoplasma cynos C142] Other Aliases:MCYN_0850 Annotation: NC_019949.1 (951497 . . . 955216, complement) ID:14356531 SEQ ID NO: 161 101. SaSA20_0769 CRISPR-associated protein[Streptococcus agalactiae SA20-06] Other Aliases: SaSA20_0769 Genomiccontext: Chromosome Annotation: NC_019048.1 (803597 . . . 807709) ID:13908026 SEQ ID NO: 162 102. csn1 CRISPR-associated protein, Csn1 family[Streptococcus pyogenes A20] Other Aliases: A20_0810 Genomic context:Chromosome Annotation: NC_018936.1 (772038 . . . 776144) ID: 13864445SEQ ID NO: 163 103. P700755_000291 CRISPR-associated protein Cas9/Csn1,subtype II [Psychroflexus torquis ATCC 700755] Other Aliases:P700755_000291 Genomic context: Chromosome Annotation: NC_018721.1(312899 . . . 317428) ID: 13804571 SEQ ID NO: 164 104. A911_07335CRISPR-associated protein [Campylobacter jejuni subsp. jejuni PT14]Other Aliases: A911_07335 Genomic context: Chromosome Annotation:NC_018709.2 (1450217 . . . 1453180, complement) ID: 13791138 SEQ ID NO:165 105. ASU2_02495 CRISPR-associated endonuclease Csn1 family protein[Actinobacillus suis H91-0380] Other Aliases: ASU2_02495 Genomiccontext: Chromosome Annotation: NC_018690.1 (552318 . . . 555482) ID:13751600 SEQ ID NO: 166 106. csn1 CRISPR-associated protein [Listeriamonocytogenes SLCC2540] Other Aliases: LMOSLCC2540_2635 Annotation:NC_018586.1 (2700744 . . . 2704748, complement) ID: 13647248 SEQ ID NO:167 107. csn1 CRISPR-associated protein [Listeria monocytogenesSLCC5850] Other Aliases: LMOSLCC5850_2605 Annotation: NC_018592.1(2646023 . . . 2650027, complement) ID: 13626042 SEQ ID NO: 168 108.csn1 CRISPR-associated protein [Listeria monocytogenes serotype 7 str.SLCC2482] Other Aliases: LMOSLCC2482_2606 Annotation: NC_018591.1(2665393 . . . 2669397, complement) ID: 13605045 SEQ ID NO: 169 109.csn1 CRISPR-associated protein [Listeria monocytogenes SLCC2755] OtherAliases: LMOSLCC2755_2607 Annotation: NC_018587.1 (2694850 . . .2698854, complement) ID: 13599053 SEQ ID NO: 170 110. BN148_1523cCRISPR-associated protein [Campylobacter jejuni subsp. jejuni NCTC11168-BN148] Other Aliases: BN148_1523c Annotation: NC_018521.1 (1456880. . . 1459834, complement) ID: 13530688 SEQ ID NO: 171 111. Belba_3201CRISPR-associated protein Cas9/Csn1, subtype II/NMEMI [Belliella balticaDSM 15883] Other Aliases: Belba_3201 Genomic context: ChromosomeAnnotation: NC_018010.1 (3445311 . . . 3449369, complement) ID: 13056967SEQ ID NO: 172 112. FN3523_1121 membrane protein [Francisella cf.novicida 3523] Other Aliases: FN3523_1121 Genomic context: ChromosomeAnnotation: NC_017449.1 (1129528 . . . 1134468, complement) ID: 12924881SEQ ID NO: 173 113. cas9 CRISPR-associated protein Cas9/Csn1, subtypeII/NMEMI [Prevotella intermedia 17] Other Aliases: PIN17_A0201Chromosome: II Annotation: Chromosome IINC_017861.1 (240722 . . .244864) ID: 12849954 SEQ ID NO: 174 114. csn1 CRISPR-associated protein,Csn1 family [Streptococcus thermophilus JIM 8232] Other Aliases:STH8232_0853 Annotation: NC_017581.1 (706443 . . . 709808) ID: 12637306SEQ ID NO: 175 115. LMOG_01918 CRISPR-associated protein [Listeriamonocytogenes J0161] Other Aliases: LMOG_01918 Genomic context:Chromosome Annotation: NC_017545.1 (2735374 . . . 2739378, complement)ID: 12557915 SEQ ID NO: 176 116. LMRG_02138 CRISPR-associated protein[Listeria monocytogenes 10403S] Other Aliases: LMRG_02138 Genomiccontext: Chromosome Annotation: NC_017544.1 (2641981 . . . 2645985,complement) ID: 12554876 SEQ ID NO: 177 117. CJSA_1443 putativeCRISPR-associated protein [Campylobacter jejuni subsp. jejuni IA3902]Other Aliases: CJSA_1443 Genomic context: Chromosome Annotation:NC_017279.1 (1454273 . . . 1457227, complement) ID: 12250720 SEQ ID NO:178 118. csn1 CRISPR-associated protein Csn1 [Streptococcus pyogenesMGAS1882] Other Aliases: MGAS1882_0792 Genomic context: ChromosomeAnnotation: NC_017053.1 (775696 . . . 779799) ID: 12014080 SEQ ID NO:179 119. csn1 CRISPR-associated protein Csn1 [Streptococcus pyogenesMGAS15252] Other Aliases: MGAS15252_0796 Genomic context: ChromosomeAnnotation: NC_017040.1 (778271 . . . 782374) ID: 11991096 SEQ ID NO:180 120. cas3 CRISPR-associated endonuclease [Corynebacteriumdiphtheriae HC02] Other Aliases: CDHC02_0036 Genomic context: ChromosomeAnnotation: NC_016802.1 (37125 . . . 40379) ID: 11739116 SEQ ID NO: 181121. cas3 CRISPR-associated endonuclease [Corynebacterium diphtheriae C7(beta)] Other Aliases: CDC7B_0035 Genomic context: ChromosomeAnnotation: NC_016801.1 (36309 . . . 39563) ID: 11737358 SEQ ID NO: 182122. cas3 CRISPR-associated endonuclease [Corynebacterium diphtheriaeBH8] Other Aliases: CDBH8_0038 Genomic context: Chromosome Annotation:NC_016800.1 (37261 . . . 40515) ID: 11735325 SEQ ID NO: 183 123. cas3CRISPR-associated endonuclease [Corynebacterium diphtheriae 31A] OtherAliases: CD31A_0036 Genomic context: Chromosome Annotation: NC_016799.1(34597 . . . 37851) ID: 11731168 SEQ ID NO: 184 124. cas3CRISPR-associated endonuclease [Corynebacterium diphtheriae VA01] OtherAliases: CDVA01_0033 Genomic context: Chromosome Annotation: NC_016790.1(34795 . . . 38049) ID: 11717708 SEQ ID NO: 185 125. cas3CRISPR-associated endonuclease [Corynebacterium diphtheriae HC01] OtherAliases: CDHC01_0034 Genomic context: Chromosome Annotation: NC_016786.1(35060 . . . 38314) ID: 11708318 SEQ ID NO: 186 126. cas9CRISPR-associated protein [Corynebacterium diphtheriae HC01] OtherAliases: CDHC01_2103 Genomic context: Chromosome Annotation: NC_016786.1(2246368 . . . 2248998) ID: 11708126 SEQ ID NO: 187 127. PARA_18570hypothetical protein [Haemophilus parainfluenzae T3T1] Other Aliases:PARA_18570 Genomic context: Chromosome Annotation: NC_015964.1 (1913335. . . 1916493) ID: 11115627 SEQ ID NO: 188 128. HDN1F_34120 hypotheticalprotein [gamma proteobacterium HdN1] Other Aliases: HDN1F_34120 Genomiccontext: Chromosome Annotation: NC_014366.1 (4143336 . . . 4146413,complement) ID: 9702142 SEQ ID NO: 189 129. SPy_1046 hypotheticalprotein [Streptococcus pyogenes M1 GAS] Other Aliases: SPy_1046 Genomiccontext: Chromosome Annotation: NC_002737.1 (854757 . . . 858863) ID:901176 SEQ ID NO: 190 130. GBS222_0765 Hypothetical protein[Streptococcus agalactiae] Other Aliases: GBS222_0765 Annotation:NC_021195.1 (810875 . . . 814987) ID: 15484689 SEQ ID NO: 191 131.NE061598_03330 hypothetical protein [Francisella tularensis subsp.tularensis NE061598] Other Aliases: NE061598_03330 Genomic context:Chromosome Annotation: NC_017453.1 (601219 . . . 604590) ID: 12437259SEQ ID NO: 192 132. NMV_1993 hypothetical protein [Neisseriameningitidis 8013] Other Aliases: NMV_1993 Annotation: NC_017501.1(1917073 . . . 1920321) ID: 12393700 SEQ ID NO: 193 133. csn1hypothetical protein [Campylobacter jejuni subsp. jejuni M1] OtherAliases: CJM1_1467 Genomic context: Chromosome Annotation: NC_017280.1(1433667 . . . 1436252, complement) ID: 12249021 SEQ ID NO: 194 134.FTU_0629 hypothetical protein [Francisella tularensis subsp. tularensisTIGB03] Other Aliases: FTU_0629 Genomic context: Chromosome Annotation:NC_016933.1 (677092 . . . 680463) ID: 11890131 SEQ ID NO: 195 135.NMAA_0315 hypothetical protein [Neisseria meningitidis WUE 2594] OtherAliases: NMAA_0315 Annotation: NC_017512.1 (377010 . . . 380258,complement) ID: 12407849 SEQ ID NO: 196 136. WS1445 hypothetical protein[Wolinella succinogenes DSM 1740] Other Aliases: WS1445 Genomic context:Chromosome Annotation: NC_005090.1 (1388202 . . . 1391381, complement)ID: 2554690 SEQ ID NO: 197 137. THITE_2123823 hypothetical protein[Thielavia terrestris NRRL 8126] Other Aliases: THITE_2123823Chromosome: 6 Annotation: Chromosome 6NC_016462.1 (1725696 . . .1725928) ID: 11523019 SEQ ID NO: 198 138. XAC29_16635 hypotheticalprotein [Xanthomonas axonopodis Xac29-1] Other Aliases: XAC29_16635Genomic context: Chromosome Annotation: NC_020800.1 (3849847 . . .3850302) ID: 14853997 SEQ ID NO: 199 139. M1GAS476_0830 hypotheticalprotein [Streptococcus pyogenes M1 476] Other Aliases: M1GAS476_0830Chromosome: 1 Annotation: NC_020540.1 (792119 . . . 796225) ID: 14819166SEQ ID NO: 200 140. Piso0_000203 Piso0_000203 [Millerozyma farinosa CBS7064] Other Aliases: GNLVRS01_PISO0A04202g Other Designations:hypothetical protein Chromosome: A Annotation: NC_020226.1 (343553 . . .343774, complement) ID: 14528449 SEQ ID NO: 201 141. G148_0828hypothetical protein [Riemerella anatipestifer RA-CH-2] Other Aliases:G148_0828 Genomic context: Chromosome Annotation: NC_020125.1 (865673 .. . 869875) ID: 14447195 SEQ ID NO: 202 142. csn1 hypothetical protein[Streptococcus dysgalactiae subsp. equisimilis AC-2713] Other Aliases:SDSE_1207 Annotation: NC_019042.1 (1134173 . . . 1138288, complement)ID: 13901498 SEQ ID NO: 203 143. A964_0899 hypothetical protein[Streptococcus agalactiae GD201008-001] Other Aliases: A964_0899 Genomiccontext: Chromosome Annotation: NC_018646.1 (935164 . . . 939276) ID:13681619 SEQ ID NO: 204 144. FNFX1_0762 hypothetical protein[Francisella cf. novicida Fx1] Other Aliases: FNFX1_0762 Genomiccontext: Chromosome Annotation: NC_017450.1 (781484 . . . 786373) ID:12435564 SEQ ID NO: 205 145. FTV_0545 hypothetical protein [Francisellatularensis subsp. tularensis TI0902] Other Aliases: FTV_0545 Genomiccontext: Chromosome Annotation: NC_016937.1 (601185 . . . 604556) ID:11880693 SEQ ID NO: 206 146. FTL_1327 hypothetical protein [Francisellatularensis subsp. holarctica LVS] Other Aliases: FTL_1327 Genomiccontext: Chromosome Annotation: NC_007880.1 (1262508 . . . 1263689,complement) ID: 3952607 SEQ ID NO: 207 147. FTL_1326 hypotheticalprotein [Francisella tularensis subsp. holarctica LVS] Other Aliases:FTL_1326 Genomic context: Chromosome Annotation: NC_007880.1 (1261927 .. . 1262403, complement) ID: 3952606 SEQ ID NO: 208

1. An in vitro method for modifying a genome at a genomic locus ofinterest in a mouse ES cell, the method comprising: contacting the mouseES cell with: a Cas9 protein; a CRISPR RNA that hybridizes to a CRISPRtarget sequence at the genomic locus of interest; a tracrRNA; and anincoming nucleic acid sequence that is flanked by: (i) a 5′ homology armthat is homologous to a 5′ target sequence at the genomic locus ofinterest; and (ii) a 3′ homolog arm that is homologous to a 3′ targetsequence at the genomic locus of interest; wherein the incoming nucleicacid sequence is at least 20 kb in size; wherein following thecontacting step, the genome of the mouse ES cell is modified to comprisea targeted genetic modification comprising: deletion of a region of thegenomic locus of interest wherein the deletion is at least 20 kb; and/orinsertion of the insert nucleic acid at the genomic locus of interestwherein the insertion is at least 20 kb. wherein the targeted genomicmodification comprises insertion of: a. One or more human antibody heavychain variable domains; b. One or more human antibody kappa light chainvariable domains; or c. One or more human antibody lambda light chainvariable domains;
 2. The method of claim 1, wherein the targeted genomicmodification comprises deletion of one or more mouse antibody heavychain variable domains and insertion of one or more human antibody heavychain variable domains.
 3. The method of claim 1, wherein the targetedgenomic modification comprises deletion of one or more mouse antibodykappa light chain variable domains and insertion of one or more humanantibody kappa light chain variable domains.
 4. The method of claim 1,wherein the targeted genomic modification comprises deletion of one ormore mouse antibody lambda light chain variable domains and insertion ofone or more human antibody lambda light chain variable domains.
 5. Themethod of claim 1, wherein the mouse ES cell or progeny thereof isdeveloped into a mouse.
 6. A mouse obtained by the method of claim
 1. 7.The mouse of claim 6, wherein the mouse is heterozygous for the targetedgenomic modification.
 8. The mouse of claim 6, wherein the mouse ishomozygous for the targeted genomic modification.
 9. The mouse of claim6, wherein the mouse further comprises a targeted modification to insertone or more human antibody kappa light chain variable domains.
 10. Themouse of claim 6, wherein the mouse further comprises a homozygoustargeted modification to insert one or more human antibody kappa lightchain variable domains.
 11. An antibody produced by the mouse of claim6.
 12. The antibody according to claim 11, wherein the mouse isheterozygous for the targeted genomic modification.
 13. The antibodyaccording to claim 11, wherein the mouse is homozygous for the targetedgenomic modification
 14. The antibody according to claim 11, wherein themouse further comprises a targeted modification to insert one or morehuman antibody kappa light chain variable domains.
 15. The antibodyaccording to claim 11, wherein the mouse further comprises a homozygoustargeted modification to insert one or more human antibody kappa lightchain variable domains.
 16. A method for producing an antibodycomprising: producing a mouse with a modified genome from the ES cellmodified by the method of claim 1 or a progeny thereof, immunizing themouse with an antigen, and isolating an antibody produced by the mouse.17. The method of claim 16, wherein the targeted genomic modificationcomprises deletion of one or more mouse antibody heavy chain variabledomains and insertion of one or more human antibody heavy chain variabledomains
 18. The method of claim 16, wherein the targeted genomicmodification comprises deletion of one or more mouse antibody kappalight chain variable domains and insertion of one or more human antibodykappa light chain variable domains.
 19. The method of claim 16, whereinthe targeted genomic modification comprises deletion of one or moremouse antibody lambda light chain variable domains and insertion of oneor more human antibody lambda light chain variable domains.